Nucleic acid detection employing charged adducts

ABSTRACT

The present invention relates to means for the detection and characterization of nucleic acid sequences, as well as variations in nucleic acid sequences. The present invention also relates to methods for forming a nucleic acid cleavage structure on a target sequence and cleaving the nucleic acid cleavage structure in a site-specific manner. The 5′ nuclease activity of a variety of enzymes is used to cleave the target-dependent cleavage structure, thereby indicating the presence of specific nucleic acid sequences or specific variations thereof. The present invention further relates to methods and devices for the separation of nucleic acid molecules based by charge.

[0001] This is a Continuation-In-Part of co-pending application Ser. No.08/599,491, filed on Jan. 24, 1996.

FIELD OF THE INVENTION

[0002] The present invention relates to means for the detection andcharacterization of nucleic acid sequences and variations in nucleicacid sequences. The present invention relates to methods for forming anucleic acid cleavage structure on a target sequence and cleaving thenucleic acid cleavage structure in a site-specific manner. The 5′nuclease activity of a variety of enzymes is used to cleave thetarget-dependent cleavage structure, thereby indicating the presence ofspecific nucleic acid sequences or specific variations thereof. Thepresent invention further provides novel methods and devices for theseparation of nucleic acid molecules based by charge.

BACKGROUND OF THE INVENTION

[0003] The detection and characterization of specific nucleic acidsequences and sequence variations has been utilized to detect thepresence of viral or bacterial nucleic acid sequences indicative of aninfection, the presence of variants or alleles of mammalian genesassociated with disease and cancers and the identification of the sourceof nucleic acids found in forensic samples, as well as in paternitydeterminations.

[0004] Various methods are known to the art which may be used to detectand characterize specific nucleic acid sequences and sequence variants.Nonetheless, as nucleic acid sequence data of the human genome, as wellas the genomes of pathogenic organisms accumulates, the demand for fast,reliable, cost-effective and user-friendly tests for the detection ofspecific nucleic acid sequences continues to grow. Importantly, thesetests must be able to create a detectable signal from samples whichcontain very few copies of the sequence of interest. The followingdiscussion examines two levels of nucleic acid detection assayscurrently in use: I. Signal Amplification Technology for detection ofrare sequences; and II. Direct Detection Technology for detection ofhigher copy number sequences.

[0005] I. Signal Amplification Technology Methods for Amplification

[0006] The “Polymerase Chain Reaction” (PCR) comprises the firstgeneration of methods for nucleic acid amplification. However, severalother methods have been developed that employ the same basis ofspecificity, but create signal by different amplification mechanisms.These methods include the “Ligase Chain Reaction” (LCR), “Self-SustainedSynthetic Reaction” (3SR/NASBA), and “Qβ-Replicase” (Qβ).

[0007] Polymerase Chain Reaction (PCR)

[0008] The polymerase chain reaction (PCR), as described in U.S. Pat.Nos. 4,683,195 and 4,683,202 to Mullis and Mullis et al. (thedisclosures of which are hereby incorporated by reference), describe amethod for increasing the concentration of a segment of target sequencein a mixture of genomic DNA without cloning or purification. Thistechnology provides one approach to the problems of low target sequenceconcentration. PCR can be used to directly increase the concentration ofthe target to an easily detectable level. This process for amplifyingthe target sequence involves introducing a molar excess of twooligonucleotide primers which are complementary to their respectivestrands of the double-stranded target sequence to the DNA mixturecontaining the desired target sequence. The mixture is denatured andthen allowed to hybridize. Following hybridization, the primers areextended with polymerase so as to form complementary strands. The stepsof denaturation, hybridization, and polymerase extension can be repeatedas often as needed, in order to obtain relatively high concentrations ofa segment of the desired target sequence.

[0009] The length of the segment of the desired target sequence isdetermined by the relative positions of the primers with respect to eachother, and, therefore, this length is a controllable parameter. Becausethe desired segments of the target sequence become the dominantsequences (in terms of concentration) in the mixture, they are said tobe “PCR-amplified.”

[0010] Ligase Chain Reaction (LCR or LAR)

[0011] The ligase chain reaction (LCR; sometimes referred to as “LigaseAmplification Reaction” (LAR) described by Barany, Proc. Natl. Acad.Sci., 88:189 (1991); Barany, PCR Methods and Applic., 1:5 (1991); and Wuand Wallace, Genomics 4:560 (1989) has developed into a well-recognizedalternative method for amplifying nucleic acids. In LCR, fouroligonucleotides, two adjacent oligonucleotides which uniquely hybridizeto one strand of target DNA, and a complementary set of adjacentoligonucleotides, which hybridize to the opposite strand are mixed andDNA ligase is added to the mixture. Provided that there is completecomplementarity at the junction, ligase will covalently link each set ofhybridized molecules. Importantly, in LCR, two probes are ligatedtogether only when they base-pair with sequences in the target sample,without gaps or mismatches. Repeated cycles of denaturation,hybridization and ligation amplify a short segment of DNA. LCR has alsobeen used in combination with PCR to achieve enhanced detection ofsingle-base changes. Segev, PCT Public. No. WO9001069 μl (1990).However, because the four oligonucleotides used in this assay can pairto form two short ligatable fragments, there is the potential for thegeneration of target-independent background signal. The use of LCR formutant screening is limited to the examination of specific nucleic acidpositions.

[0012] Self-Sustained Synthetic Reaction (3SR/NASBA)

[0013] The self-sustained sequence replication reaction (3SR) (Guatelliet al., Proc. Natl. Acad. Sci., 87:1874-1878 [1990], with an erratum atProc. Natl. Acad. Sci., 87:7797 [1990]) is a transcription-based invitro amplification system (Kwok et al., Proc. Natl. Acad. Sci.,86:1173-1177 [1989]) that can exponentially amplify RNA sequences at auniform temperature. The amplified RNA can then be utilized for mutationdetection (Fahy et al., PCR Meth. Appl., 1:25-33 [1991]). In thismethod, an oligonucleotide primer is used to add a phage RNA polymerasepromoter to the 5′ end of the sequence of interest. In a cocktail ofenzymes and substrates that includes a second primer, reversetranscriptase, RNase H, RNA polymerase and ribo-and deoxyribonucleosidetriphosphates, the target sequence undergoes repeated rounds oftranscription, cDNA synthesis and second-strand synthesis to amplify thearea of interest. The use of 3SR to detect mutations is kineticallylimited to screening small segments of DNA (e.g., 200-300 base pairs).

[0014] Q-Beta (Qβ) Replicase

[0015] In this method, a probe which recognizes the sequence of interestis attached to the replicatable RNA template for Qβ replicase. Apreviously identified major problem with false positives resulting fromthe replication of unhybridized probes has been addressed through use ofa sequence-specific ligation step. However, available thermostable DNAligases are not effective on this RNA substrate, so the ligation must beperformed by T4 DNA ligase at low temperatures (37° C.). This preventsthe use of high temperature as a means of achieving specificity as inthe LCR, the ligation event can be used to detect a mutation at thejunction site, but not elsewhere.

[0016] Table 1 below, lists some of the features desirable for systemsuseful in sensitive nucleic acid diagnostics, and summarizes theabilities of each of the major amplification methods (See also,Landgren, Trends in Genetics 9:199 [1993]).

[0017] A successful diagnostic method must be very specific. Astraight-forward method of controlling the specificity of nucleic acidhybridization is by controlling the temperature of the reaction. Whilethe 3SR/NASBA, and Qβ systems are all able to generate a large quantityof signal, one or more of the enzymes involved in each cannot be used athigh temperature (i.e., >55° C.). Therefore the reaction temperaturescannot be raised to prevent non-specific hybridization of the probes. Ifprobes are shortened in order to make them melt more easily at lowtemperatures, the likelihood of having more than one perfect match in acomplex genome increases. For these reasons, PCR and LCR currentlydominate the research field in detection technologies. TABLE 1 METHOD:PCR & 3SR FEATURE PCR LCR LCR NASBA Qβ Amplifies Target + + + +Recognition of Independent + + + + + Sequences Required Performed atHigh Temp. + + Operates at Fixed Temp. + + ExponentialAmplification + + + + + Generic Signal Generation + Easily Automatable

[0018] The basis of the amplification procedure in the PCR and LCR isthe fact that the products of one cycle become usable templates in allsubsequent cycles, consequently doubling the population with each cycle.The final yield of any such doubling system can be expressed as:(1+X)=y, where “X” is the mean efficiency (percent copied in eachcycle), “n” is the number of cycles, and “y” is the overall efficiency,or yield of the reaction (Mullis, PCR Methods Applic., 1:1 [1991]). Ifevery copy of a target DNA is utilized as a template in every cycle of apolymerase chain reaction, then the mean efficiency is 100%. If 20cycles of PCR are performed, then the yield will be 2²⁰ , or 1,048,576copies of the starting material. If the reaction conditions reduce themean efficiency to 85%, then the yield in those 20 cycles will be only1.85²⁰, or 220,513 copies of the starting material. In other words, aPCR running at 85% efficiency will yield only 21% as much final product,compared to a reaction running at 100% efficiency. A reaction that isreduced to 50% mean efficiency will yield less than 1% of the possibleproduct.

[0019] In practice, routine polymerase chain reactions rarely achievethe theoretical maximum yield, and PCRs are usually run for more than 20cycles to compensate for the lower yield. At 50% mean efficiency, itwould take 34 cycles to achieve the million-fold amplificationtheoretically possible in 20, and at lower efficiencies, the number ofcycles required becomes prohibitive. In addition, any backgroundproducts that amplify with a better mean efficiency than the intendedtarget will become the dominant products.

[0020] Also, many variables can influence the mean efficiency of PCR,including target DNA length and secondary structure, primer length anddesign, primer and dNTP concentrations, and buffer composition, to namebut a few. Contamination of the reaction with exogenous DNA (e.g., DNAspilled onto lab surfaces) or cross-contamination is also a majorconsideration. Reaction conditions must be carefully optimized for eachdifferent primer pair and target sequence, and the process can takedays, even for an experienced investigator. The laboriousness of thisprocess, including numerous technical considerations and other factors,presents a significant drawback to using PCR in the clinical setting.Indeed, PCR has yet to penetrate the clinical market in a significantway. The same concerns arise with LCR, as LCR must also be optimized touse different oligonucleotide sequences for each target sequence. Inaddition, both methods require expensive equipment, capable of precisetemperature cycling.

[0021] Many applications of nucleic acid detection technologies, such asin studies of allelic variation, involve not only detection of aspecific sequence in a complex background, but also the discriminationbetween sequences with few, or single, nucleotide differences. Onemethod for the detection of allele-specific variants by PCR is basedupon the fact that it is difficult for Taq polymerase to synthesize aDNA strand when there is a mismatch between the template strand and the3′ end of the primer. An allele-specific variant may be detected by theuse of a primer that is perfectly matched with only one of the possiblealleles; the mismatch to the other allele acts to prevent the extensionof the primer, thereby preventing the amplification of that sequence.This method has a substantial limitation in that the base composition ofthe mismatch influences the ability to prevent extension across themismatch, and certain mismatches do not prevent extension or have only aminimal effect (Kwok et al., Nucl. Acids Res., 18:999 [1990]).)

[0022] A similar 3′-mismatch strategy is used with greater effect toprevent ligation in the LCR (Barany, PCR Meth. Applic., 1:5 [1991]). Anymismatch effectively blocks the action of the thermostable ligase, butLCR still has the drawback of target-independent background ligationproducts initiating the amplification. Moreover, the combination of PCRwith subsequent LCR to identify the nucleotides at individual positionsis also a clearly cumbersome proposition for the clinical laboratory.

[0023] II. Direct Detection Technology

[0024] When a sufficient amount of a nucleic acid to be detected isavailable, there are advantages to detecting that sequence directly,instead of making more copies of that target, (e.g., as in PCR and LCR).Most notably, a method that does not amplify the signal exponentially ismore amenable to quantitative analysis. Even if the signal is enhancedby attaching multiple dyes to a single oligonucleotide, the correlationbetween the final signal intensity and amount of target is direct. Sucha system has an additional advantage that the products of the reactionwill not themselves promote further reaction, so contamination of labsurfaces by the products is not as much of a concern. Traditionalmethods of direct detection including Northern and Southern blotting andRNase protection assays usually require the use of radioactivity and arenot amenable to automation. Recently devised techniques have sought toeliminate the use of radioactivity and/or improve the sensitivity inautomatable formats. Two examples are the “Cycling Probe Reaction”(CPR), and “Branched DNA” (bDNA)

[0025] The cycling probe reaction (CPR) (Duck et al., BioTech., 9:142[1990]), uses a long chimeric oligonucleotide in which a central portionis made of RNA while the two termini are made of DNA. Hybridization ofthe probe to a target DNA and exposure to a thermostable RNase H causesthe RNA portion to be digested. This destabilizes the remaining DNAportions of the duplex, releasing the remainder of the probe from thetarget DNA and allowing another probe molecule to repeat the process.The signal, in the form of cleaved probe molecules, accumulates at alinear rate. While the repeating process increases the signal, the RNAportion of the oligonucleotide is vulnerable to RNases that may carriedthrough sample preparation.

[0026] Branched DNA (bDNA), described by Urdea et al., Gene 61:253-264(1987), involves oligonucleotides with branched structures that alloweach individual oligonucleotide to carry 35 to 40 labels (e.g., alkalinephosphatase enzymes). While this enhances the signal from ahybridization event, signal from non-specific binding is similarlyincreased.

[0027] While both of these methods have the advantages of directdetection discussed above, neither the CPR or bDNA methods can make useof the specificity allowed by the requirement of independent recognitionby two or more probe (oligonucleotide) sequences, as is common in thesignal amplification methods described in section I. above. Therequirement that two oligonucleotides must hybridize to a target nucleicacid in order for a detectable signal to be generated confers an extrameasure of stringency on any detection assay. Requiring twooligonucleotides to bind to a target nucleic acid reduces the chancethat false “positive” results will be produced due to the non-specificbinding of a probe to the target. The further requirement that the twooligonucleotides must bind in a specific orientation relative to thetarget, as is required in PCR, where oligonucleotides must be oppositelybut appropriately oriented such that the DNA polymerase can bridge thegap between the two oligonucleotides in both directions, furtherenhances specificity of the detection reaction. However, it is wellknown to those in the art that even though PCR utilizes twooligonucleotide probes (termed primers) “non-specific” amplification(i.e., amplification of sequences not directed by the two primers used)is a common artifact. This is in part because the DNA polymerase used inPCR can accommodate very large distances, measured in nucleotides,between the oligonucleotides and thus there is a large window in whichnon-specific binding of an oligonucleotide can lead to exponentialamplification of inappropriate product. The LCR, in contrast, cannotproceed unless the oligonucleotides used are bound to the targetadjacent to each other and so the full benefit of the dualoligonucleotide hybridization is realized.

[0028] An ideal direct detection method would combine the advantages ofthe direct detection assays (e.g., easy quantification and minimal riskof carry-over contamination) with the specificity provided by a dualoligonucleotide hybridization assay.

SUMMARY OF THE INVENTION

[0029] The present invention relates to means for cleaving a nucleicacid cleavage structure in a site-specific manner. In one embodiment,the means for cleaving is a cleaving enzyme comprising 5′ nucleasesderived from thermostable DNA polymerases. These polymerases form thebasis of a novel method of detection of specific nucleic acid sequences.The present invention contemplates use of novel detection methods forvarious uses, including, but not limited to clinical diagnosticpurposes.

[0030] In one embodiment, the present invention contemplates a DNAsequence encoding a DNA polymerase altered in sequence (i.e., a “mutant”DNA polymerase) relative to the native sequence, such that it exhibitsaltered DNA synthetic activity from that of the native (i.e., “wildtype”) DNA polymerase. It is preferred that the encoded DNA polymeraseis altered such that it exhibits reduced synthetic activity compared tothat of the native DNA polymerase. In this manner, the enzymes of theinvention are predominantly 5′ nucleases and are capable of cleavingnucleic acids in a structure-specific manner in the absence ofinterfering synthetic activity.

[0031] Importantly, the 5′ nucleases of the present invention arecapable of cleaving linear duplex structures to create single discretecleavage products. These linear structures are either 1) not cleaved bythe wild type enzymes (to any significant degree), or 2) are cleaved bythe wild type enzymes so as to create multiple products. Thischaracteristic of the 5′ nucleases has been found to be a consistentproperty of enzymes derived in this manner from thermostable polymerasesacross eubacterial thermophilic species.

[0032] It is not intended that the invention be limited by the nature ofthe alteration necessary to render the polymerase synthesis-deficient.Nor is it intended that the invention be limited by the extent of thedeficiency. The present invention contemplates various structures,including altered structures (primary, secondary, etc.), as well asnative structures, that may be inhibited by synthesis inhibitors.

[0033] Where the polymerase structure is altered, it is not intendedthat the invention be limited by the means by which the structure isaltered. In one embodiment, the alteration of the native DNA sequencecomprises a change in a single nucleotide. In another embodiment, thealteration of the native DNA sequence comprises a deletion of one ormore nucleotides. In yet another embodiment, the alteration of thenative DNA sequence comprises an insertion of one or more nucleotides.It is contemplated that the change in DNA sequence may manifest itselfas change in amino acid sequence.

[0034] The present invention contemplates 5′ nucleases from a variety ofsources, including mesophilic, psychrophilic, thermophilic, andhyperthermophilic organisms. The preferred 5′ nucleases arethermostable. Thermostable 5′ nucleases are contemplated as particularlyuseful in that they operate at temperatures where nucleic acidhybridization is extremely specific, allowing for allele-specificdetection (including single-base mismatches). In one embodiment, thethermostable 5′ nucleases are selected from the group consisting ofaltered polymerases derived from the native polymerases of Thermusspecies, including, but not limited to Thermus aquaticus, Thermusflavus, and Thermus thermophilus. However, the invention is not limitedto the use of thermostable 5′ nucleases.

[0035] As noted above, the present invention contemplates the use ofaltered polymerases in a detection method. In one embodiment, thepresent invention provides a method of detecting the presence of atarget RNA by detecting non-target cleavage products comprising: a)providing: i) a cleavage means, ii) a source of target RNA, where thetarget RNA has a first region, a second region and a third region,wherein the first region is located adjacent to and downstream from thesecond region, and the second region is located adjacent to anddownstream from the third region, iii) a first oligonucleotide having a5′ and a 3′ portion, wherein the 5′ portion of the first oligonucleotidecontains a sequence complementary to the second region of the target RNAand wherein the 3′ portion of the first oligonucleotide contains asequence complementary to the third region of the target RNA, iv) asecond oligonucleotide having a 5′ and a 3′ portion wherein the 5′portion of the second oligonucleotide contains a sequence complementaryto the first region of the target RNA, and the 3′ portion of the secondoligonucleotide contains a sequence complementary to the second regionof the target RNA; b) mixing the cleavage means, the target RNA, and thefirst and second oligonucleotides, to create a reaction mixture underreaction conditions such that at least the 3′ portion of the firstoligonucleotide is annealed to the target RNA, and wherein at least the5′ portion of the second oligonucleotide is annealed to the target RNAso as to create a cleavage structure, and wherein cleavage of thecleavage structure occurs to generate non-target cleavage products; andc) detecting the non-target cleavage products.

[0036] It is contemplated that the first, second and third regions ofthe target be located adjacent to each other. However, the invention isnot limited to the use of a target in which the three regions arecontiguous with each other. Thus, the present invention contemplates theuse of target RNAs wherein these three regions are contiguous with eachother, as well as target RNAs wherein these three regions are notcontiguous. It is further contemplated that gaps of approximately 2-10nucleotides, representing regions of non-complementarity to theoligonucleotides (e.g., the first and/or second oligonucleotides), maybe present between the three regions of the target RNA.

[0037] In at least one embodiment, it is intended that mixing of step b)is conducted under conditions such that at least the 3′ portion of thefirst oligonucleotide is annealed to the target RNA, and wherein atleast the 5′ portion of the second oligonucleotide is annealed to thetarget RNA. In this manner a cleavage structure is created and cleavageof this cleavage structure can occur. These conditions allow for the useof various formats. In a preferred format, the conditions of mixingcomprises mixing together the target RNA with the first and secondoligonucleotides and the cleavage means in an aqueous solution in whicha source of divalent cations is lacking. In this format, the cleavagereaction is initiated by the addition of a solution containing Mn²⁺ orMg²⁺ ions. In another preferred format, the conditions of mixingcomprises mixing together the target RNA, and the first and secondoligonucleotides in an aqueous solution containing Mn²⁺ or Mg²⁺ ions,and then adding the cleavage means to the reaction mixture.

[0038] The invention is not limited by the means employed for thedetection of the non-target cleavage products. For example, the productsgenerated by the cleavage reaction (i.e., the non-target cleavageproducts) may be detected by their separation of the reaction productson agarose or polyacrylamide gels and staining with ethidium bromide.Other non-gel-based detection methods are provided herein.

[0039] It is contemplated that the oligonucleotides may be labelled.Thus, if the cleavage reaction employs a first oligonucleotidecontaining a label, detection of the non-target cleavage products maycomprise detection of the label. The invention is not limited by thenature of the label chosen, including, but not limited to, labels whichcomprise a dye or a radionucleotide (e.g. ³²P), fluorescein moiety, abiotin moiety, luminogenic, fluorogenic, phosphorescent, or fluors incombination with moieties that can suppress emission by fluorescenceenergy transfer (FET). Numerous methods are available for the detectionof nucleic acids containing any of the above-listed labels. For example,biotin-labeled oligonucleotide(s) may be detected using non-isotopicdetection methods which employ streptavidin-alkaline phosphataseconjugates. Fluorescein-labelled oligonucleotide(s) may be detectedusing a fluorescein-imager.

[0040] It is also contemplated that labelled oligonucleotides (cleavedor uncleaved) may be separated by means other than electrophoresis. Forexample, biotin-labelled oligonucleotides may be separated from nucleicacid present in the reaction mixture using para-magnetic or magneticbeads, or particles which are coated with avidin (or streptavidin). Inthis manner, the biotinylated oligonucleotide/avidin-magnetic beadcomplex can be physically separated from the other components in themixture by exposing the complexes to a magnetic field. Additionally, thesignal from the cleaved oligonucleotides may be resolved from that ofthe uncleaved oligonucleotides without physical separation. For example,a change in size, and therefore rate of rotation in solution offluorescent molecules can be detected by fluorescence polarizationanalysis.

[0041] In a preferred embodiment, the reaction conditions comprise acleavage reaction temperature which is less than the melting temperatureof the first oligonucleotide and greater than the melting temperature ofthe 3′ portion of the first oligonucleotide. In a particularly preferredembodiment, the reaction temperature is between approximately 40-65° C.It is contemplated that the reaction temperature at which the cleavagereaction occurs be selected with regard to the guidelines provided inthe Description of the Invention.

[0042] The invention is not limited by the nature of theoligonucleotides employed. Using a target RNA, the oligonucleotides maycomprise DNA, RNA or an oligonucleotide comprising a mixture of RNA andDNA.

[0043] The invention also contemplates the use of a secondoligonucleotide (i.e., the upstream oligonucleotide) which comprises afunctional group (e.g., a 5′ peptide region) which prevents thedissociation of the 5′ portion of the second oligonucleotide from thefirst region of the target RNA. When such a functional group is presenton the second oligonucleotide, the interaction between the 3′ portion ofthe second oligonucleotide and the first region of the target RNA may bedestabilized (i.e., designed to have a lower local melting temperature)through the use of A-T (or A-U) rich sequences, base analogs that formfewer hydrogen bonds (e.g., dG-dU pairs) or through the use ofphosphorothioate backbones, in order to allow the 5′ region of the firstoligonucleotide to compete successfully for hybridization.

[0044] In a preferred embodiment, the cleavage means comprises athermostable 5′ nuclease. The thermostable 5′ nuclease may have aportion of the amino acid sequence that is homologous to a portion ofthe amino acid sequence of a thermostable DNA polymerase derived from athermophilic organism. It is contemplated that thermophilic organismswill be selected from such species as those within the genus Thermus,including, but not limited to Thermus aquaticus, Thermus flavus andThermus thermophilus. Preferred nucleases are encoded by DNA sequencesselected from the group consisting of SEQ ID NOS:1-3, 9, 10, 12, 21, 30and 31.

[0045] In one embodiment, the present invention contemplates a DNAsequence encoding a DNA polymerase altered in sequence (i.e., a “mutant”DNA polymerase) relative to the native sequence, such that it exhibitsaltered DNA synthetic activity from that of the native (i.e., “wildtype”) DNA polymerase. With regard to the polymerase, a complete absenceof synthesis is not required. However, it is desired that cleavagereactions occur in the absence of polymerase activity at a level thatinterferes with the method. It is preferred that the encoded DNApolymerase is altered such that it exhibits reduced synthetic activityfrom that of the native DNA polymerase. In this manner, the enzymes ofthe invention are nucleases and are capable of cleaving nucleic acids ina structure-specific manner. Importantly, the nucleases of the presentinvention are capable of cleaving cleavage structures to create discretecleavage products.

[0046] The present invention utilizes such enzymes in methods fordetection and characterization of nucleic acid sequences and sequencechanges. The present invention also relates to means for cleaving anucleic acid cleavage structure in a site-specific manner. Nucleaseactivity is used to screen for known and unknown mutations, includingsingle base changes, in nucleic acids.

[0047] The invention is not limited to use of oligonucleotides which arecompletely complementary to their cognate target sequences. In oneembodiment, both the first and second oligonucleotides are completelycomplementary to the target RNA. In another embodiment, the firstoligonucleotide is partially complementary to the target RNA. In yetanother embodiment, the second oligonucleotide is partiallycomplementary to the target RNA. In yet another embodiment, both thefirst and the second oligonucleotide are partially complementary to thetarget RNA.

[0048] In a preferred embodiment, the methods of the invention employ asource of target RNA which comprises a sample selected from the groupincluding, but not limited to blood, saliva, cerebral spinal fluid,pleural fluid, milk, lymph, sputum and semen.

[0049] In a preferred embodiment, the method employs reaction conditionswhich comprise providing a source of divalent cations. In a particularlypreferred embodiment, the divalent cation is selected from the groupcomprising Mn²⁺ and Mg²⁺ ions.

[0050] The novel detection methods of the invention may be employed forthe detection of target RNAs including, but not limited to, target RNAscomprising wild type and mutant alleles of genes, including genes fromhumans or other animals that are or may be associated with disease orcancer. In addition, the methods of the invention may be used for thedetection of and/or identification of strains of microorganisms,including bacteria, fungi, protozoa, ciliates and viruses (and inparticular for the detection and identification of RNA viruses, such asHCV).

[0051] The present invention further provides a method of separatingnucleic acid molecules, comprising: a) providing: i) a charge-balancedoligonucleotide and ii) a reactant; b) mixing the charge-balancedoligonucleotide with the reactant to create a reaction mixture underconditions such that a charge-unbalanced oligonucleotide is produced;and c) separating the charge-unbalanced oligonucleotide from thereaction mixture.

[0052] The method of the present invention is not limited by the natureof the reactant employed. In a preferred embodiment the reactantcomprises a cleavage means. In a particularly preferred embodiment, thecleavage means is an endonuclease. In another embodiment, the cleavagemeans is an exonuclease. In a still further embodiment, the reactantcomprises a polymerization means. In another embodiment, the reactantcomprises a ligation means.

[0053] In a preferred embodiment, the charge-balanced oligonucleotidecomprises a label. The invention is not limited by the nature of thelabel chosen, including, but not limited to, labels which comprise a dyeor a radionucleotide (e.g., ³²P), fluorescein moiety, a biotin moiety,luminogenic, fluorogenic, phosphorescent, or fluors in combination withmoieties that can suppress emission by fluorescence energy transfer(FET). The label may be a charged moeity or alternatively may be acharge neutral moeity.

[0054] In another preferred embodiment, the charge-balancedoligonucleotide comprises one or more phosphonate groups. In a preferredembodiment, the phosphonate group is a methylphosphonate group.

[0055] In one embodiment, the charge-balanced oligonucleotide has a netneutral charge and the charge-unbalanced oligonucleotide has a netpositive charge. Alternatively, the charge-balanced oligonucleotide hasa net neutral charge and the charge-unbalanced oligonucleotide has a netnegative charge. In yet another alternative embodiment, thecharge-balanced oligonucleotide has a net negative charge and thecharge-unbalanced oligonucleotide has a net positive charge. In anotherembodiment, the charge-balanced oligonucleotide has a net negativecharge and the charge-unbalanced oligonucleotide has a net neutralcharge. In another preferred embodiment, the charge-balancedoligonucleotide has a net positive charge and the charge-unbalancedoligonucleotide has a net neutral charge. Still further, thecharge-balanced oligonucleotide has a net positive charge and thecharge-unbalanced oligonucleotide has a net negative charge.

[0056] In a preferred embodiment, the charge-balanced oligonucleotidecomprises DNA containing one or more positively charged adducts. In apreferred embodiment, the charge-balanced oligonucleotide comprises DNAcontaining one or more positively charged adducts and the cleavage meansremoves one or more nucleotides from the charge-balanced oligonucleotideto produce the charge-unbalanced oligonucleotide, wherein thecharge-unbalanced oligonucleotide has a net positive charge. In anotherpreferred embodiment, the charge-balanced oligonucleotide comprises DNAcontaining one or more positively charged adducts and the cleavage meansremoves one or more nucleotides from the charge-balanced oligonucleotideto produce the charge-unbalanced oligonucleotide, wherein thecharge-unbalanced oligonucleotide has a net neutral charge. Stillfurther, the charge-balanced oligonucleotide comprises DNA containingone or more positively charged adducts and the cleavage means removesone or more nucleotides from the charge-balanced oligonucleotide toproduce the charge-unbalanced oligonucleotide, wherein thecharge-unbalanced oligonucleotide has a net negative charge.

[0057] In a preferred embodiment, the charge-balanced oligonucleotidecomprises DNA containing one or more negatively charged adducts (e.g.,negatively charged amino acids). Examples of negative charged adductsinclude negatively charged amino acids (e.g., aspartate and glutamate).In a preferred embodiment, the charge-balanced oligonucleotide comprisesDNA containing one or more negatively charged adducts and the cleavagemeans removes one or more nucleotides from the charge-balancedoligonucleotide to produce the charge-unbalanced oligonucleotide,wherein the charge-unbalanced oligonucleotide has a net negative charge.In a preferred embodiment, the charge-balanced oligonucteotide comprisesDNA containing one or more negatively charged adducts and the cleavagemeans removes one or more nucleotides from the charge-balancedoligonucleotide to produce the charge-unbalanced oligonucleotide,wherein the charge-unbalanced oligonucleotide has a net neutral charge.In a preferred embodiment, the charge-balanced oligonucleotide comprisesDNA containing one or more negatively charged adducts and the cleavagemeans removes one or more nucleotides from the charge-balancedoligonucleotide to produce the charge-unbalanced oligonucleotide,wherein the charge-unbalanced oligonucleotide has a net negative charge.

[0058] The present invention is not limited by the nature of thepositively charged adduct(s) employed. In a preferred embodiment, thepositively charged adducts are selected from the group consisting ofindodicarbocyanine dye amidites (e.g., Cy3 and Cy5), amino-substitutednucleotides, ethidium bromide, ethidium homodimer,(1,3-propanediamino)propidium, (diethylenetriamino)propidium, thiazoleorange, (N-N′-tetramethyl-1,3-propanediamino)propyl thiazole orange,(N-N′-tetramethyl-1,2-ethanediamino)propyl thiazole orange, thiazoleorange-thiazole orange homodimer (TOTO), thiazole orande-thiazole blueheterodimer (TOTAB), thiazole orange-ethidium heterodimer 1 (TOED1),thiazole orange-ethidium heterodimer 2 (TOED2), florescien-ethidiumheterodimer (FED) and positively charged amino acids.

[0059] In another preferred embodiment, the separating step comprisessubjecting the reaction mixture to an electrical field comprising apositive pole and a negative pole under conditions such that thecharge-unbalanced oligonucleotide migrates toward the positive pole(i.e., electrode). In another embodiment, the separating step comprisessubjecting the reaction mixture to an electrical field comprising apositive pole and a negative pole under conditions such that thecharge-unbalanced oligonucleotide migrates toward the negative pole.

[0060] In still further embodiment, the method of the present inventionfurther comprises detecting the presence of the separatedcharge-unbalanced oligonucleotide. The present invetion is not limitedby the detection method employed; the method of detection chosen willvary depending on the nature of the label employed (if one is employed).

[0061] The present invention further comprises a method of detectingcleaved nucleic molecules, comprising: a) providing: i) a homogeneousplurality of charge-balanced oligonucleotides; ii) a sample suspected ofcontaining a target nucleic acid having a sequence comprising a firstregion complementary to said charge-balanced oligonucleotide; iii) acleavage means; and iv) a reaction vessel; b) adding to said vessel, inany order, the sample, the charge-balanced oligonucleotides and thecleavage means to create a reaction mixture under conditions such that aportion of the charge-balanced oligonucleotides binds to thecomplementary target nucleic acid to create a bound (i.e., annealed)population, and such that the cleavage means cleaves at least a portionof said bound population of charge-balanced oligonucleotides to producea population of unbound, charge-unbalanced oligonucleotides; and c)separating the unbound, charge-unbalanced oligonucleotides from thereaction mixture.

[0062] In a preferred embodiment, the method further comprises providinga homogeneous plurality of oligonucleotides complementary to a secondregion of the target nucleic acid, wherein the oligonucleotides arecapable of binding to the target nucleic acid upstream of thecharge-balanced oligonucleotides. In another preferred embodiment, thefirst and the second region of the target nucleic acid share a region ofoverlap.

[0063] The invention is not limited by the nature of the clevage meansemployed. In one embodiment, the cleavage means comprises a thermostable5′ nuclease. In a preferred embodiment, a portion of the amino acidsequence of the 5′ nuclease is homologous to a portion of the amino acidsequence of a thermostable DNA polymerase derived from a thermophilicorganism. In a preferred embodiment, the organism is selected from thegroup consisting of Thermus aquaticus, Thermus flavus and Thermusthermophilus. In another preferred embodiment, the nuclease is encodedby a DNA sequence selected from the group consisting of SEQ ID NOS:1-3,9, 10, 12, 21, 30 and 31.

[0064] The invention is not limited by the nature of the target nucleicacid. The target nucleic acid may comprise single-stranded DNA,double-stranded DNA or RNA. In a preferred embodiment, the targetnucleic acid comprises double-stranded DNA and prior to the addition ofthe cleavage means the reaction mixture is treated such that thedouble-stranded DNA is rendered substantially single-stranded preferablyby increasing the temperature.

[0065] The invention further provides a method of separating nucleicacid molecules, comprising: a) modifying an oligonucleotide so as toproduce a charge-balanced oligonucleotide; b) providing: i) a saidcharge-balanced oligonucleotide and ii) a reactant; c) mixing saidcharge-balanced oligonucleotide with said reactant to create a reactionmixture under conditions such that a charge-unbalanced oligonucleotideis produced; and d) separating said charge-unbalanced oligonucleotidefrom said reaction mixture.

[0066] The invention is not limited by the nature of the modification.In a preferred embodiment, the modifying step comprises the covalentattachment of a positively charged adduct to one or bases of theoligonucleotide. In another preferred embodiment, the modifying stepcomprises the covalent attachment of a negatively charged adduct to oneor bases of the oligonucleotide. In a still further embodiment, themodifying comprises the incorporation of one or more amino-substitutedbases during synthesis of the oligonucleotide. In another embodiment,the modifying comprises the incorporation of one or more phosphonategroups during synthesis of said oligonucleotide. In a preferredembodiment, the phosphonate group is a methylphosphonate group.

[0067] The invention further provides a method of treating a nucleicacid molecule, comprising: a) providing: i) a charge-balancedoligonucleotide and ii) a reactant; b) mixing said charge-balancedoligonucleotide with said reactant to create a reaction mixture underconditions such that a charge-unbalanced oligonucleotide is produced.

[0068] The invention further provides a method of treating a nucleicacid molecule, comprising: a) modifying an oligonucleotide so as toproduce a charge-balanced oligonucleotide; b) providing: i) saidcharge-balanced oligonucleotide and ii) a reactant; c) mixing thecharge-balanced oligonucleotide with the reactant to create a reactionmixture under conditions such that a charge-unbalanced oligonucleotideis produced.

DESCRIPTION OF THE DRAWINGS

[0069]FIG. 1A provides a schematic of one embodiment of the detectionmethod of the present invention.

[0070]FIG. 1B provides a schematic of a second embodiment of thedetection method of the present invention.

[0071]FIG. 2 is a comparison of the nucleotide structure of the DNAPgenes isolated from Thermus aquaticus (SEQ ID NO:1), Thermus flavus (SEQID NO:2) and Thermus thermophilus (SEQ ID NO:3); the consensus sequence(SEQ ID NO:7) is shown at the top of each row.

[0072]FIG. 3 is a comparison of the amino acid sequence of the DNAPisolated from Thermus aquaticus (SEQ ID NO:4), Thermus flavus (SEQ IDNO:5), and Thermus thermophilus (SEQ ID NO:6); the consensus sequence(SEQ ID NO:8) is shown at the top of each row.

[0073] FIGS. 4A-G are a set of diagrams of wild-type andsynthesis-deficient DNAPTaq genes.

[0074]FIG. 5A depicts the wild-type Thermus flavus polymerase gene.

[0075]FIG. 5B depicts a synthesis-deficient Thermus flavus polymerasegene.

[0076]FIG. 6 depicts a structure which cannot be amplified usingDNAPTaq.

[0077]FIG. 7 is a ethidium bromide-stained gel demonstrating attempts toamplify a bifurcated duplex using either DNAPTaq or DNAPStf (i.e., theStoffel fragment of DNAPTaq).

[0078]FIG. 8 is an autoradiogram of a gel analyzing the cleavage of abifurcated duplex by DNAPTaq and lack of cleavage by DNAPStf.

[0079] FIGS. 9A-B are a set of autoradiograms of gels analyzing cleavageor lack of cleavage upon addition of different reaction components andchange of incubation temperature during attempts to cleave a bifurcatedduplex with DNAPTaq.

[0080] FIGS. 10A-B are an autoradiogram displaying timed cleavagereactions, with and without primer.

[0081] FIGS. 11A-B are a set of autoradiograms of gels demonstratingattempts to cleave a bifurcated duplex (with and without primer) withvarious DNAPs.

[0082]FIGS. 12A shows the substrates and oligonucleotides used to testthe specific cleavage of substrate DNAs targeted by pilotoligonucleotides.

[0083]FIG. 12B shows an autoradiogram of a gel showing the results ofcleavage reactions using the substrates and oligonucleotides shown FIG.12A.

[0084]FIG. 13A shows the substrate and oligonucleotide used to test thespecific cleavage of a substrate RNA targeted by a pilotoligonucleotide.

[0085]FIG. 13B shows an autoradiogram of a gel showing the results of acleavage reaction using the substrate and oligonucleotide shown in FIG.13A.

[0086]FIG. 14 is a diagram of vector pTTQ18.

[0087]FIG. 15 is a diagram of Vector pET-3c.

[0088] FIGS. 16A-E depicts a set of molecules which are suitablesubstrates for cleavage by the 5′ nuclease activity of DNAPs.

[0089]FIG. 17 is an autoradiogram of a gel showing the results of acleavage reaction run with synthesis-deficient DNAPs.

[0090]FIG. 18 is an autoradiogram of a PEI chromatogram resolving theproducts of an assay for synthetic activity in synthesis-deficientDNAPTaq clones.

[0091]FIG. 19A depicts the substrate molecule used to test the abilityof synthesis-deficient DNAPs to cleave short hairpin structures.

[0092]FIG. 19B shows an autoradiogram of a gel resolving the products ofa cleavage reaction run using the substrate shown in FIG. 19A.

[0093]FIG. 20A shows the A- and T-hairpin molecules used in thetrigger/detection assay.

[0094]FIG. 20B shows the sequence of the alpha primer used in thetrigger/detection assay.

[0095]FIG. 20C shows the structure of the cleaved A- and T-hairpinmolecules.

[0096]FIG. 20D depicts the complementarity between the A- and T-hairpinmolecules.

[0097]FIG. 21 provides the complete 206-mer duplex sequence employed asa substrate for the 5′ nucleases of the present invention

[0098]FIGS. 22A and B show the cleavage of linear nucleic acidsubstrates (based on the 206-mer of FIG. 21) by wild type DNAPs and 5′nucleases isolated from Thermus aquaticus and Thermus flavus.

[0099]FIG. 23 provides a detailed schematic corresponding to the of oneembodiment of the detection method of the present invention.

[0100]FIG. 24 shows the propagation of cleavage of the linear duplexnucleic acid structures of FIG. 23 by the 5′ nucleases of the presentinvention.

[0101]FIG. 25A shows the “nibbling” phenomenon detected with the DNAPsof the present invention.

[0102]FIG. 25B shows that the “nibbling” of FIG. 25A is 5′ nucleolyticcleavage and not phosphatase cleavage.

[0103]FIG. 26 demonstrates that the “nibbling” phenomenon is duplexdependent.

[0104]FIG. 27 is a schematic showing how “nibbling” can be employed in adetection assay.

[0105]FIG. 28 demonstrates that “nibbling” can be target directed.

[0106]FIG. 29 provides a schematic drawing of a target nucleic acid withan invader oligonucleotide and a probe oligonucleotide annealed to thetarget.

[0107]FIG. 30 provides a schematic showing the S-60 hairpinoligonucleotide (SEQ ID NO:40) with the annealed P-15 oligonucletide(SEQ ID NO:41).

[0108]FIG. 31 is an autoradiogram of a gel showing the results of acleavage reaction run using the S-60 hairpin in the presence or absenceof the P-15 oligonucleotide.

[0109]FIG. 32 provides a schematic showing three different arrangementsof target-specific oligonucleotides and their hybridization to a targetnucleic acid which also has a probe oligonucleotide annealed thereto.

[0110]FIG. 33 is the image generated by a fluorescence imager showingthat the presence of an invader oligonucleotide causes a shift in thesite of cleavage in a probe/target duplex.

[0111]FIG. 34 is the image generated by a fluorescence imager showingthe products of invader-directed cleavage assays run using the threetarget-specific oligonucleotides diagrammed in FIG. 32.

[0112]FIG. 35 is the image generated by a fluorescence imager showingthe products of invader-directed cleavage assays run in the presence orabsence of non-target nucleic acid molecules.

[0113]FIG. 36 is the image generated by a fluorescence imager showingthe products of invader-directed cleavage assays run in the presence ofdecreasing amounts of target nucleic acid.

[0114]FIG. 37 is the image generated by a fluorescence imager showingthe products of invader-directed cleavage assays run in the presence orabsence of saliva extract using various thermostable 5′ nucleases or DNApolymerases.

[0115]FIG. 38 is the image generated by a fluorescence imager showingthe products of invader-directed cleavage assays run using various 5′nucleases.

[0116]FIG. 39 is the image generated by a fluorescence imager showingthe products of invader-directed cleavage assays run using two targetnucleic acids which differ by a single basepair at two differentreaction temperatures.

[0117]FIG. 40A provides a schematic showing the effect of elevatedtemperature upon the annealing and cleavage of a probe oligonucleotidealong a target nucleic acid wherein the probe contains a region ofnoncomplementarity with the target.

[0118]FIG. 40B provides a schematic showing the effect of adding anupstream oligonucleotide upon the annealing and cleavage of a probeoligonucleotide along a target nucleic acid wherein the probe contains aregion of noncomplementarity with the target.

[0119]FIG. 41 provides a schematic showing an arrangement of atarget-specific invader oligonucleotide (SEQ ID NO:50) and atarget-specific probe oligonucleotide (SEQ ID NO:49) bearing a 5′ Cy3label along a target nucleic acid (SEQ ID NO:42).

[0120]FIG. 42 is the image generated by a fluorescence imager showingthe products of invader-directed cleavage assays run in the presence ofincreasing concentrations of KCl.

[0121]FIG. 43 is the image generated by a fluorescence imager showingthe products of invader-directed cleavage assays run in the presence ofincreasing concentrations of NaCl.

[0122]FIG. 44 is the image generated by a fluorescence imager showingthe products of invader-directed cleavage assays run in the presence ofincreasing concentrations of LiCl.

[0123]FIG. 45 is the image generated by a fluorescence imager showingthe products of invader-directed cleavage assays run in the presence ofincreasing concentrations of KGlu.

[0124]FIG. 46 is the image generated by a fluorescence imager showingthe products of invader-directed cleavage assays run in the presence ofincreasing concentrations of MnCl₂ or MgCl₂.

[0125]FIG. 47 is the image generated by a fluorescence imager showingthe products of invader-directed cleavage assays run in the presence ofincreasing concentrations of CTAB.

[0126]FIG. 48 is the image generated by a fluorescence imager showingthe products of invader-directed cleavage assays run in the presence ofincreasing concentrations of PEG.

[0127]FIG. 49 is the image generated by a fluorescence imager showingthe products of invader-directed cleavage assays run in the presence ofglycerol, Tween-20 and/or Nonidet-P40.

[0128]FIG. 50 is the image generated by a fluorescence imager showingthe products of invader-directed cleavage assays run in the presence ofincreasing concentrations of gelatin in reactions containing or lackingKCl or LiCl.

[0129]FIG. 51 is the image generated by a fluorescence imager showingthe products of invader-directed cleavage assays run in the presence ofincreasing amounts of genomic DNA or tRNA.

[0130]FIG. 52 is the image generated by a fluorescence imager showingthe products of invader-directed cleavage assays run use a HCV RNAtarget.

[0131]FIG. 53 is the image generated by a fluorescence imager showingthe products of invader-directed cleavage assays run using a HCV RNAtarget and demonstrate the stability of RNA targets underinvader-directed cleavage assay conditions.

[0132]FIG. 54 is the image generated by a fluorescence imager showingthe sensitivity of detection and the stability of RNA ininvader-directed cleavage assays run using a HCV RNA target.

[0133]FIG. 55 is the image generated by a fluorescence imager showingthermal degradation of oligonucleotides containing or lacking a 3′phosphate group.

[0134]FIG. 56 depicts the structure of amino-modified oligonucleotides70 and 74.

[0135]FIG. 57 depicts the structure of amino-modified oligonucleotide 75

[0136]FIG. 58 depicts the structure of amino-modified oligonucteotide76.

[0137]FIG. 59 is the image generated by a fluorescence imager scan of anIEF gel showing the migration of substrates 70, 70dp, 74, 74dp, 75,75dp, 76 and 76dp.

[0138]FIG. 60A provides a schematic showing an arrangement of atarget-specific invader oligonucleotide (SEQ ID NO:61) and atarget-specific probe oligonucleotide (SEQ ID NO:62) bearing a 5′ Cy3label along a target nucleic acid (SEQ ID NO:63).

[0139]FIG. 60B is the image generated by a fluorescence imager showingthe detection of specific cleavage products generated in an invasivecleavage assay using charge reversal (i.e., charge based separation ofcleavage products).

[0140]FIG. 61 is the image generated by a fluorescence imager whichdepicts the sensitivity of detection of specific cleavage productsgenerated in an invasive cleavage assay using charge reversal.

[0141]FIG. 62 depicts a first embodiment of a device for thecharge-based separation of oligonucleotides.

[0142]FIG. 63 depicts a second embodiment of a device for thecharge-based separation of oligonucleotides.

[0143]FIG. 64 shows an autoradiogram of a gel showing the results ofcleavage reactions run in the presence or absence of a primeroligonucleotide; a sequencing ladder is shown as a size marker.

[0144]FIGS. 65a-d depict four pairs of oligonucleotides; in each pairshown, the upper arrangement of a probe annealed to a target nucleicacid lacks an upstream oligonucleotide and the lower arrangementcontains an upstream oligonucleotide.

[0145]FIG. 66 shows the chemical structure of several positively chargedheterodimeric DNA-binding dyes.

DEFINITIONS

[0146] As used herein, the terms “complementary” or “complementarity”are used in reference to polynucleotides (i.e., a sequence ofnucleotides such as an oligonucleotide or a target nucleic acid) relatedby the base-pairing rules. For example, for the sequence “A-G-T,” iscomplementary to the sequence “T-C-A.” Complementarity may be “partial,”in which only some of the nucleic acids' bases are matched according tothe base pairing rules. Or, there may be “complete” or “total”complementarity between the nucleic acids. The degree of complementaritybetween nucleic acid strands has significant effects on the efficiencyand strength of hybridization between nucleic acid strands. This is ofparticular importance in amplification reactions, as well as detectionmethods which depend upon binding between nucleic acids.

[0147] The term “homology” refers to a degree of identity. There may bepartial homology or complete homology. A partially identical sequence isone that is less than 100% identical to another sequence.

[0148] As used herein, the term “hybridization” is used in reference tothe pairing of complementary nucleic acids. Hybridization and thestrength of hybridization (i.e., the strength of the association betweenthe nucleic acids) is impacted by such factors as the degree ofcomplementary between the nucleic acids, stringency of the conditionsinvolved, the T_(m) of the formed hybrid, and the G:C ratio within thenucleic acids.

[0149] As used herein, the term “T_(m)” is used in reference to the“melting temperature.” The melting temperature is the temperature atwhich a population of double-stranded nucleic acid molecules becomeshalf dissociated into single strands. The equation for calculating theTm of nucleic acids is well known in the art. As indicated by standardreferences, a simple estimate of the Tm value may be calculated by theequation: T_(m)=81.5+0.41(% G+C), when a nucleic acid is in aqueoussolution at 1 M NaCl (see e.g., Anderson and Young, Quantitative FilterHybridization, in Nucleic Acid Hybridization (1985). Other referencesinclude more sophisticated computations which take structural as well assequence characteristics into account for the calculation of T_(m).

[0150] As used herein the term “stringency” is used in reference to theconditions of temperature, ionic strength, and the presence of othercompounds, under which nucleic acid hybridizations are conducted. With“high stringency” conditions, nucleic acid base pairing will occur onlybetween nucleic acid fragments that have a high frequency ofcomplementary base sequences. Thus, conditions of “weak” or “low”stringency are often required when it is desired that nucleic acidswhich are not completely complementary to one another be hybridized orannealed together.

[0151] The term “gene” refers to a DNA sequence that comprises controland coding sequences necessary for the production of a polypeptide orprecursor. The polypeptide can be encoded by a full length codingsequence or by any portion of the coding sequence so long as the desiredenzymatic activity is retained.

[0152] The term “wild-type” refers to a gene or gene product which hasthe characteristics of that gene or gene product when isolated from anaturally occurring source. A wild-type gene is that which is mostfrequently observed in a population and is thus arbitrarily designed the“normal” or “wild-type” form of the gene. In contrast, the term“modified” or “mutant” refers to a gene or gene product which displaysmodifications in sequence and or functional properties (L e., alteredcharacteristics) when compared to the wild-type gene or gene product. Itis noted that naturally-occurring mutants can be isolated; these areidentified by the fact that they have altered characteristics whencompared to the wild-type gene or gene product.

[0153] The term “recombinant DNA vector” as used herein refers to DNAsequences containing a desired coding sequence and appropriate DNAsequences necessary for the expression of the operably linked codingsequence in a particular host organism. DNA sequences necessary forexpression in procaryotes include a promoter, optionally an operatorsequence, a ribosome binding site and possibly other sequences.Eukaryotic cells are known to utilize promoters, polyadenlyation signalsand enhancers.

[0154] The term “LTR” as used herein refers to the long terminal repeatfound at each end of a provirus (i.e., the integrated form of aretrovirus). The LTR contains numerous regulatory signals includingtranscriptional control elements, polyadenylation signals and sequencesneeded for replication and integration of the viral genome. The viralLTR is divided into three regions called U3, R and U5.

[0155] The U3 region contains the enhancer and promoter elements. The U5region contains the polyadenylation signals. The R (repeat) regionseparates the U3 and U5 regions and transcribed sequences of the Rregion appear at both the 5′ and 3′ ends of the viral RNA.

[0156] The term “oligonucleotide” as used herein is defined as amolecule comprised of two or more deoxyribonucleotides orribonucleotides, preferably at least 5 nucleotides, more preferably atleast about 10-15 nucleotides and more preferably at least about 15 to30 nucleotides. The exact size will depend on many factors, which inturn depends on the ultimate function or use of the oligonucleotide. Theoligonucleotide may be generated in any manner, including chemicalsynthesis, DNA replication, reverse transcription, or a combinationthereof.

[0157] Because mononucleotides are reacted to make oligonucleotides in amanner such that the 5′ phosphate of one mononucleotide pentose ring isattached to the 3′ oxygen of its neighbor in one direction via aphosphodiester linkage, an end of an oligonucleotide is referred to asthe “5′ end” if its 5′ phosphate is not linked to the 3′ oxygen of amononucleotide pentose ring and as the “3′ end” if its 3′ oxygen is notlinked to a 5′ phosphate of a subsequent mononucleotide pentose ring. Asused herein, a nucleic acid sequence, even if internal to a largeroligonucleotide, also may be said to have 5′ and 3′ ends. A first regionalong a nucleic acid strand is said to be upstream of another region ifthe 3′ end of the first region is before the 5′ end of the second regionwhen moving along a strand of nucleic acid in a 5′ to 3′ direction.

[0158] When two different, non-overlapping oligonucleotides anneal todifferent regions of the same linear complementary nucleic acidsequence, and the 3′ end of one oligonucleotide points towards the 5′end of the other, the former may be called the “upstream”oligonucleotide and the latter the “downstream” oligonucleotide.

[0159] The term “primer” refers to an oligonucleotide which is capableof acting as a point of initiation of synthesis when placed underconditions in which primer extension is initiated. An oligonucleotide“primer” may occur naturally, as in a purified restriction digest or maybe produced synthetically.

[0160] A primer is selected to be “substantially” complementary to astrand of specific sequence of the template. A primer must besufficiently complementary to hybridize with a template strand forprimer elongation to occur. A primer sequence need not reflect the exactsequence of the template. For example, a non-complementary nucleotidefragment may be attached to the 5′ end of the primer, with the remainderof the primer sequence being substantially complementary to the strand.Non-complementary bases or longer sequences can be interspersed into theprimer, provided that the primer sequence has sufficient complementaritywith the sequence of the template to hybridize and thereby form atemplate primer complex for synthesis of the extension product of theprimer.

[0161] “Hybridization” methods involve the annealing of a complementarysequence to the target nucleic acid (the sequence to be detected; thedetection of this sequence may be by either direct or indirect means).The ability of two polymers of nucleic acid containing complementarysequences to find each other and anneal through base pairing interactionis a well-recognized phenomenon. The initial observations of the“hybridization” process by Marmur and Lane, Proc. Natl. Acad. Sci. USA46:453 (1960) and Doty et al., Proc. Natl. Acad. Sci. USA 46:461 (1960)have been followed by the refinement of this process into an essentialtool of modern biology.

[0162] With regard to complementarity, it is important for somediagnostic applications to determine whether the hybridizationrepresents complete or partial complementarity. For example, where it isdesired to detect simply the presence or absence of pathogen DNA (suchas from a virus, bacterium, fungi, mycoplasma, protozoan) it is onlyimportant that the hybridization method ensures hybridization when therelevant sequence is present; conditions can be selected where bothpartially complementary probes and completely complementary probes willhybridize. Other diagnostic applications, however, may require that thehybridization method distinguish between partial and completecomplementarity. It may be of interest to detect genetic polymorphisms.For example, human hemoglobin is composed, in part, of four polypeptidechains. Two of these chains are identical chains of 141 amino acids(alpha chains) and two of these chains are identical chains of 146 aminoacids (beta chains). The gene encoding the beta chain is known toexhibit polymorphism. The normal allele encodes a beta chain havingglutamic acid at the sixth position. The mutant allele encodes a betachain having valine at the sixth position. This difference in aminoacids has a profound (most profound when the individual is homozygousfor the mutant allele) physiological impact known clinically as sicklecell anemia. It is well known that the genetic basis of the amino acidchange involves a single base difference between the normal allele DNAsequence and the mutant allele DNA sequence.

[0163] The complement of a nucleic acid sequence as used herein refersto an oligonucleotide which, when aligned with the nucleic acid sequencesuch that the 5′ end of one sequence is paired with the 3′ end of theother, is in “antiparallel association.” Certain bases not commonlyfound in natural nucleic acids may be included in the nucleic acids ofthe present invention and include, for example, inosine and7-deazaguanine. Complementarity need not be perfect; stable duplexes maycontain mismatched base pairs or unmatched bases. Those skilled in theart of nucleic acid technology can determine duplex stabilityempirically considering a number of variables including, for example,the length of the oligonucleotide, base composition and sequence of theoligonucleotide, ionic strength and incidence of mismatched base pairs.

[0164] Stability of a nucleic acid duplex is measured by the meltingtemperature, or “T_(m).” The T_(m) of a particular nucleic acid duplexunder specified conditions is the temperature at which on average halfof the base pairs have disassociated.

[0165] The term “label” as used herein refers to any atom or moleculewhich can be used to provide a detectable (preferably quantifiable)signal, and which can be attached to a nucleic acid or protein. Labelsmay provide signals detectable by fluorescence, radioactivity,colorimetry, gravimetry, X-ray diffraction or absorption, magnetism,enzymatic activity, and the like. A label may be a charged moeity(positive or negative charge) or alternatively, may be charge neutral.

[0166] The term “cleavage structure” as used herein, refers to astructure which is formed by the interaction of a probe oligonucleotideand a target nucleic acid to form a duplex, said resulting structurebeing cleavable by a cleavage means, including but not limited to anenzyme. The cleavage structure is a substrate for specific cleavage bysaid cleavage means in contrast to a nucleic acid molecule which is asubstrate for non-specific cleavage by agents such as phosphodiesteraseswhich cleave nucleic acid molecules without regard to secondarystructure (i.e., no formation of a duplexed structure is required).

[0167] The term “cleavage means” as used herein refers to any meanswhich is capable of cleaving a cleavage structure, including but notlimited to enzymes. The cleavage means may include native DNAPs having5′ nuclease activity (e.g., Taq DNA polymerase, E. coli DNA polymeraseI) and, more specifically, modified DNAPs having 5′ nuclease but lackingsynthetic activity. The ability of 5′ nucleases to cleave naturallyoccurring structures in nucleic acid templates (structure-specificcleavage) is useful to detect internal sequence differences in nucleicacids without prior knowledge of the specific sequence of the nucleicacid. In this manner, they are structure-specific enzymes.Structure-specific enzymes are enzymes which recognize specificsecondary structures in a nucleic molecule and cleave these structures.The cleavage means of the invention cleave a nucleic acid molecule inresponse to the formation of cleavage structures; it is not necessarythat the cleavage means cleave the cleavage structure at any particularlocation within the cleavage structure.

[0168] The cleavage means is not restricted to enzymes having solely 5′nuclease activity. The cleavage means may include nuclease activityprovided from a variety of sources including the Cleavase® enzymes, TaqDNA polymerase and E. coli DNA polymerase I.

[0169] The term “thermostable” when used in reference to an enzyme, suchas a 5′ nuclease, indicates that the enzyme is functional or active(i.e., can perform catalysis) at an elevated temperature, i.e., at about55° C. or higher.

[0170] The term “cleavage products” as used herein, refers to productsgenerated by the reaction of a cleavage means with a cleavage structure(i e., the treatment of a cleavage structure with a cleavage means).

[0171] The term “target nucleic acid” refers to a nucleic acid moleculewhich contains a sequence which has at least partial complementaritywith at least a probe oligonucleotide and may also have at least partialcomplementarity with an invader oligonucleotide. The target nucleic acidmay comprise single- or double-stranded DNA or RNA.

[0172] The term “probe oligonucleotide” refers to an oligonucleotidewhich interacts with a target nucleic acid to form a cleavage structurein the presence or absence of an invader oligonucleotide. When annealedto the target nucleic acid, the probe oligonucleotide and target form acleavage structure and cleavage occurs within the probe oligonucleotide.In the presence of an invader oligonucleotide upstream of the probeoligonucleotide along the target nucleic acid will shift the site ofcleavage within the probe oligonucleotide (relative to the site ofcleavage in the absence of the invader).

[0173] The term “non-target cleavage product” refers to a product of acleavage reaction which is not derived from the target nucleic acid. Asdiscussed above, in the methods of the present invention, cleavage ofthe cleavage structure occurs within the probe oligonucleotide. Thefragments of the probe oligonucleotide generated by this target nucleicacid-dependent cleavage are “non-target cleavage products.” The term“invader oligonucleotide” refers to an oligonucleotide which containssequences at its 3′ end which are substantially the same as sequenceslocated at the 5′ end of a probe oligonucleotide; these regions willcompete for hybridization to the same segment along a complementarytarget nucleic acid.

[0174] The term “substantially single-stranded” when used in referenceto a nucleic acid substrate means that the substrate molecule existsprimarily as a single strand of nucleic acid in contrast to adouble-stranded substrate which exists as two strands of nucleic acidwhich are held together by inter-strand base pairing interactions.

[0175] The term “sequence variation” as used herein refers todifferences in nucleic acid sequence between two nucleic acids. Forexample, a wild-type structural gene and a mutant form of this wild-typestructural gene may vary in sequence by the presence of single basesubstitutions and/or deletions or insertions of one or more nucleotides.These two forms of the structural gene are said to vary in sequence fromone another. A second mutant form of the structural gene may exist. Thissecond mutant form is said to vary in sequence from both the wild-typegene and the first mutant form of the gene.

[0176] The term “liberating” as used herein refers to the release of anucleic acid fragment from a larger nucleic acid fragment, such as anoligonucleotide, by the action of a 5′ nuclease such that the releasedfragment is no longer covalently attached to the remainder of theoligonucleotide.

[0177] The term “K_(m)” as used herein refers to the Michaelis-Mentenconstant for an enzyme and is defined as the concentration of thespecific substrate at which a given enzyme yields one-half its maximumvelocity in an enzyme catalyzed reaction.

[0178] The term “nucleotide analog” as used herein refers to modified ornon-naturally occurring nucleotides such as 7-deaza purines (i.e.,7-deaza-dATP and 7-deaza-dGTP). Nucleotide analogs include base analogsand comprise modified forms of deoxyribonucleotides as well asribonucleotides.

[0179] The term “polymorphic locus” is a locus present in a populationwhich shows variation between members of the population (i.e., the mostcommon allele has a frequency of less than 0.95). In contrast, a“monomorphic locus” is a genetic locus at little or no variations seenbetween members of the population (generally taken to be a locus atwhich the most common allele exceeds a frequency of 0.95 in the genepool of the population).

[0180] The term “microorganism” as used herein means an organism toosmall to be observed with the unaided eye and includes, but is notlimited to bacteria, virus, protozoans, fungi, and ciliates.

[0181] The term “microbial gene sequences” refers to gene sequencesderived from a microorganism.

[0182] The term “bacteria” refers to any bacterial species includingeubacterial and archaebacterial species.

[0183] The term “virus” refers to obligate, ultramicroscopic,intracellular parasites incapable of autonomous replication (i.e.,replication requires the use of the host cell's machinery).

[0184] The term “multi-drug resistant” or multiple-drug resistant”refers to a microorganism which is resistant to more than one of theantibiotics or antimicrobial agents used in the treatment of saidmicroorganism.

[0185] The term “sample” in the present specification and claims is usedin its broadest sense. On the one hand it is meant to include a specimenor culture (e.g., microbiological cultures). On the other hand, it ismeant to include both biological and environmental samples.

[0186] Biological samples may be animal, including human, fluid, solid(e.g., stool) or tissue, as well as liquid and solid food and feedproducts and ingredients such as dairy items, vegetables, meat and meatby-products, and waste. Biological samples may be obtained from all ofthe various families of domestic animals, as well as feral or wildanimals, including, but not limited to, such animals as ungulates, bear,fish, lagamorphs, rodents, etc.

[0187] Environmental samples include environmental material such assurface matter, soil, water and industrial samples, as well as samplesobtained from food and dairy processing instruments, apparatus,equipment, utensils, disposable and non-disposable items. These examplesare not to be construed as limiting the sample types applicable to thepresent invention.

[0188] The term “source of target nucleic acid” refers to any samplewhich contains nucleic acids (RNA or DNA). Particularly preferredsources of target nucleic acids are biological samples including, butnot limited to blood, saliva, cerebral spinal fluid, pleural fluid,milk, lymph, sputum and semen.

[0189] An oligonucleotide is said to be present in “excess” relative toanother oligonucleotide (or target nucleic acid sequence) if thatoligonucleotide is present at a higher molar concentration that theother oligonucleotide (or target nucleic acid sequence). When anoligonucleotide such as a probe oligonucleotide is present in a cleavagereaction in excess relative to the concentration of the complementarytarget nucleic acid sequence, the reaction may be used to indicate theamount of the target nucleic acid present. Typically, when present inexcess, the probe oligonucleotide will be present at least a 100-foldmolar excess; typically at least 1 pmole of each probe oligonucleotidewould be used when the target nucleic acid sequence was present at about10 fmoles or less.

[0190] A sample “suspected of containing” a first and a second targetnucleic acid may contain either, both or neither target nucleic acidmolecule.

[0191] The term “charge-balanced” oligonucleotide refers to anolignucleotide (the input oligonucleotide in a reaction) which has beenmodified such that the modified oligonucleotide bears a charge, suchthat when the modified oligonucleotide is either cleaved (i.e.,shortened) or elongated, a resulting product bears a charge differentfrom the input oligonucleotide (the “charge-unbalanced” oligonucleotide)thereby permitting separation of the input and reacted oligonucleotideson the basis of charge. The term “charge-balanced” does not imply thatthe modified or balanced oligonucleotide has a net neutral charge(although this can be the case). Charge-balancing refers to the designand modification of an oligonucleotide such that a specific reactionproduct generated from this input oligonucleotide can be separated onthe basis of charge from the input oligonuceotide.

[0192] For example, in an invader-directed cleavage assay in which theprobe oligonucleotide bears the sequence: 5′-TTCTTTTCACCAGCGAGACGGG-3′(i.e., SEQ ID NO:61 without the modified bases) and cleavage of theprobe occurs between the second and third residues, one possiblecharge-balanced version of this oligonuceotide would be:5′-Cy3-AminoT-Amino-TCTTTTCACCAGCGAGAC GGG-3′. This modifiedoligonucleotide bears a net negative charge. After cleavage, thefollowing oligonucleotides are generated: 5′-Cy3-AminoT-Amino-T-3′ and5′-CTTTTCACCAGCGAGACGGG-3′ (residues 3-22of SEQ ID NO:61).5′-Cy3-AminoT-Amino-T-3′ bears a detectable moeity (thepositively-charged Cy3 dye) and two amino-modified bases. Theamino-modified bases and the Cy3 dye contribute positive charges inexcess of the negative charges contributed by the phosphate groups andthus the 5′-Cy3-AminoT-Amino-T-3′ oligonucleotide has a net positivecharge. The other, longer cleavage fragment, like the input probe, bearsa net negative charge. Because the 5′-Cy3-AminoT-Amino-T-3′ fragment isseparable on the basis of charge from the input probe (thecharge-balanced oligonucleotide), it is referred to as acharge-unbalanced oligonucleotide. The longer cleavage product cannot beseparated on the basis of charge from the input oligonucleotide as botholigonucleotides bear a net negative charge; thus, the longer cleavageproduct is not a charge-unbalanced oligonucleotide.

[0193] The term “net neutral charge” when used in reference to anoligonucletide, including modified oligonucleotides, indicates that thesum of the charges present (i.e, R—NH³⁺ groups on thymidines, the N3nitrogen of cytosine, presence or absence or phosphate groups, etc.)under the desired reaction conditions is essentially zero. Anoligonucletide having a net neutral charge would not migrate in anelectrical field.

[0194] The term “net positive charge” when used in reference to anoligonucletide, including modified oligonucleotides, indicates that thesum of the charges present (i.e, R—NH³⁺ groups on thymidines, the N3nitrogen of cytosine, presence or absence or phosphate groups, etc.)under the desired reaction conditions is +1 or greater. Anoligonucletide having a net positive charge would migrate toward thenegative electrode in an electrical field.

[0195] The term “net negative charge” when used in reference to anoligonucletide, including modified oligonucleotides, indicates that thesum of the charges present (i.e, R—NH³⁺ groups on thymidines, the N3nitrogen of cytosine, presence or absence or phosphate groups, etc.)under the desired reaction conditions is −1 or lower. An oligonucletidehaving a net negative charge would migrate toward the positive electrodein an electrical field.

[0196] The term “polymerization means” refers to any agent capable offacilitating the addition of nucleoside triphosphates to anoligonucleotide. Preferred polymerization means comprise DNApolymerases.

[0197] The term “ligation means” refers to any agent capable offacilitatig the ligation (i.e., the formation of a phosphodiester bondbetween a 3′-OH and a 5′-P located at the termini of two strands ofnuceic acid). Preferred ligation means comprise DNA ligases and RNAligases.

[0198] The term “reactant” is used herein in its broadest sense. Thereactant can comprise an enzymatic reactant, a chemical reactant orultraviolet light (ultraviolet light, particulary short wavelengthultraviolet light is known to break oligonucleotide chains). Any agentcapable of reacting with an oligonucleotide to either shorten (i.e.,cleave) or elongate the oligonucleotide is encompsased within the term“reactant.” The term “adduct” is used herein in its broadest sense toindicate any compound or element which can be added to anoligonucleotide. An adduct may be charged (postively or negatively) ormay be charge neutral. An adduct may be added to the oligonucleotide viacovalent or non-covalent linkages. Examples of adducts, include but arenot limited to indodicarbocyanine dye amidites, amino-substitutednucleotides, ethidium bromide, ethidium homodimer,(1,3-propanediamino)propidium, (diethylenetriamino)propidium, thiazoleorange, (N-N′-tetramethyl-1,3-propanediamino)propyl thiazole orange,(N-N′-tetramethyl-1,2-ethanediamino)propyl thiazole orange, thiazoleorange-thiazole orange homodimer (TOTO), thiazole orande-thiazole blueheterodimer (TOTAB), thiazole orange-ethidium heterodimer 1 (TOED1),thiazole orange-ethidium heterodimer 2 (TOED2) and florescien-ethidiumheterodimer (FED), psoralens, biotin, streptavidin, avidin, etc.

[0199] Where a first oligonucleotide is complementary to a region of atarget nucleic acid and a second oligonucleotide has complementary tothe same region (or a portion of this region) a “region of overlap”exists along the target nucleic acid. The degree of overlap will varydepending upon the nature of the complementarity (see, e.g., region “X”in FIG. 29 and the accompanying discussion)

DESCRIPTION OF THE INVENTION

[0200] The present invention relates to methods and compositions fortreating nucleic acid, and in particular, methods and compositions fordetection and characterization of nucleic acid sequences and sequencechanges.

[0201] The present invention relates to means for cleaving a nucleicacid cleavage structure in a site-specific manner. In particular, thepresent invention relates to a cleaving enzyme having 5′ nucleaseactivity without interfering nucleic acid synthetic ability.

[0202] This invention provides 5′ nucleases derived from thermostableDNA polymerases which exhibit altered DNA synthetic activity from thatof native thermostable DNA polymerases. The 5′ nuclease activity of thepolymerase is retained while the synthetic activity is reduced orabsent. Such 5′ nucleases are capable of catalyzing thestructure-specific cleavage of nucleic acids in the absence ofinterfering synthetic activity. The lack of synthetic activity during acleavage reaction results in nucleic acid cleavage products of uniformsize.

[0203] The novel properties of the polymerases of the invention form thebasis of a method of detecting specific nucleic acid sequences. Thismethod relies upon the amplification of the detection molecule ratherthan upon the amplification of the target sequence itself as do existingmethods of detecting specific target sequences.

[0204] DNA polymerases (DNAPs), such as those isolated from E. coli orfrom thermophilic bacteria of the genus Thermus, are enzymes thatsynthesize new DNA strands. Several of the known DNAPs containassociated nuclease activities in addition to the synthetic activity ofthe enzyme.

[0205] Some DNAPs are known to remove nucleotides from the 5′ and 3′ends of DNA chains [Kornberg, DNA Replication, W.H. Freeman and Co., SanFrancisco, pp. 127-139 (1980)]. These nuclease activities are usuallyreferred to as 5′ exonuclease and 3′ exonuclease activities,respectively. For example, the 5′ exonuclease activity located in theN-terminal domain of several DNAPs participates in the removal of RNAprimers during lagging strand synthesis during DNA replication and theremoval of damaged nucleotides during repair. Some DNAPs, such as the E.coli DNA polymerase (DNAPEc1), also have a 3′ exonuclease activityresponsible for proof-reading during DNA synthesis (Kornberg, supra).

[0206] A DNAP isolated from Thermus aquaticus, termed Taq DNA polymerase(DNAPTaq), has a 5′ exonuclease activity, but lacks a functional 3′exonucleolytic domain [Tindall and Kunkell, Biochem. 27:6008 (1988)].Derivatives of DNAPEc1 and DNAPTaq, respectively called the Klenow andStoffel fragments, lack 5′ exonuclease domains as a result of enzymaticor genetic manipulations [Brutlag et al., Biochem. Biophys. Res. Commun.37:982 (1969); Erlich et al., Science 252:1643 (1991); Setlow andKornberg, J. Biol. Chem. 247:232 (1972)].

[0207] The 5′ exonuclease activity of DNAPTaq was reported to requireconcurrent synthesis [Gelfand, PCR Technology—Principles andApplications for DNA Amplification (H. A. Erlich, Ed.), Stockton Press,New York, p. 19 (1989)]. Although mononucleotides predominate among thedigestion products of the 5′ exonucleases of DNAPTaq and DNAPEc1, shortoligonucleotides (≦12 nucleotides) can also be observed implying thatthese so-called 5′ exonucleases can function endonucleolytically[Setlow, supra; Holland et al., Proc. Natl. Acad. Sci. USA 88:7276(1991)].

[0208] In WO 92/06200, Gelfand et al. show that the preferred substrateof the 5′ exonuclease activity of the thermostable DNA polymerases isdisplaced single-stranded DNA. Hydrolysis of the phosphodiester bondoccurs between the displaced single-stranded DNA and the double-helicalDNA with the preferred exonuclease cleavage site being a phosphodiesterbond in the double helical region. Thus, the 5′ exonuclease activityusually associated with DNAPs is a structure-dependent single-strandedendonuclease and is more properly referred to as a 5′ nuclease.Exonucleases are enzymes which cleave nucleotide molecules from the endsof the nucleic acid molecule. Endonucleases, on the other hand, areenzymes which cleave the nucleic acid molecule at internal rather thanterminal sites. The nuclease activity associated with some thermostableDNA polymerases cleaves endonucleolytically but this cleavage requirescontact with the 5′ end of the molecule being cleaved. Therefore, thesenucleases are referred to as 5′ nucleases.

[0209] When a 5′ nuclease activity is associated with a eubacterial TypeA DNA polymerase, it is found in the one-third N-terminal region of theprotein as an independent functional domain. The C-terminal two-thirdsof the molecule constitute the polymerization domain which isresponsible for the synthesis of DNA. Some Type A DNA polymerases alsohave a 3′ exonuclease activity associated with the two-third C-terminalregion of the molecule.

[0210] The 5′ exonuclease activity and the polymerization activity ofDNAPs have been separated by proteolytic cleavage or geneticmanipulation of the polymerase molecule. To date thermostable DNAPs havebeen modified to remove or reduce the amount of 5′ nuclease activitywhile leaving the polymerase activity intact.

[0211] The Klenow or large proteolytic cleavage fragment of DNAPEc1contains the polymerase and 3′ exonuclease activity but lacks the 5′nuclease activity. The Stoffel fragment of DNAPTaq (DNAPStf) lacks the5′ nuclease activity due to a genetic manipulation which deleted theN-terminal 289 amino acids of the polymerase molecule [Erlich et al.,Science 252:1643 (1991)]. WO 92/06200 describes a thermostable DNAP withan altered level of 5′ to 3′ exonuclease. U.S. Pat. No. 5,108,892describes a Thermus aquaticus DNAP without a 5′ to 3′ exonuclease.However, the art of molecular biology lacks a thermostable DNApolymerase with a lessened amount of synthetic activity.

[0212] The present invention provides 5′ nucleases derived fromthermostable Type A DNA polymerases that retain 5′ nuclease activity buthave reduced or absent synthetic activity. The ability to uncouple thesynthetic activity of the enzyme from the 5′ nuclease activity provesthat the 5′ nuclease activity does not require concurrent DNA synthesisas was previously reported (Gelfand, PCR Technology, supra).

[0213] The description of the invention is divided into: I. Detection ofSpecific Nucleic Acid Sequences Using 5′ Nucleases; II. Generation of 5′Nucleases Derived From Thermostable DNA Polymerases; III. Detection ofSpecific Nucleic Acid Sequences Using 5′ Nucleases in anInvader-Directed Cleavage Assay; IV. A Comparison Of Invasive CleavageAnd Primer-Directed Cleavage; and V. Fractionation Of Specific NucleicAcids By Selective Charge Reversal.

[0214] I. Detection of Specific Nucleic Acid Sequences Using 5′Nucleases

[0215] The 5′ nucleases of the invention form the basis of a noveldetection assay for the identification of specific nucleic acidsequences. This detection system identifies the presence of specificnucleic acid sequences by requiring the annealing of two oligonucleotideprobes to two portions of the target sequence. As used herein, the term“target sequence” or “target nucleic acid sequence” refers to a specificnucleic acid sequence within a polynucleotide sequence, such as genomicDNA or RNA, which is to be either detected or cleaved or both.

[0216]FIG. 1A provides a schematic of one embodiment of the detectionmethod of the present invention. The target sequence is recognized bytwo distinct oligonucleotides in the triggering or trigger reaction. Itis preferred that one of these oligonucleotides is provided on a solidsupport. The other can be provided free. In FIG. 1A the free oligo isindicated as a “primer” and the other oligo is shown attached to a beaddesignated as type 1. The target nucleic acid aligns the twooligonucleotides for specific cleavage of the 5′ arm (of the oligo onbead 1) by the DNAPs of the present invention (not shown in FIG. 1A).

[0217] The site of cleavage (indicated by a large solid arrowhead) iscontrolled by the distance between the 3′ end of the “primer” and thedownstream fork of the oligo on bead 1. The latter is designed with anuncleavable region (indicated by the striping). In this manner neitheroligonucleotide is subject to cleavage when misaligned or whenunattached to target nucleic acid.

[0218] Successful cleavage releases a single copy of what is referred toas the alpha signal oligo. This oligo may contain a detectable moiety(e.g., fluorescein). On the other hand, it may be unlabelled.

[0219] In one embodiment of the detection method, two moreoligonucleotides are provided on solid supports. The oligonucleotideshown in FIG. 1A on bead 2 has a region that is complementary to thealpha signal oligo (indicated as alpha prime) allowing forhybridization. This structure can be cleaved by the DNAPs of the presentinvention to release the beta signal oligo. The beta signal oligo canthen hybridize to type 3 beads having an oligo with a complementaryregion (indicated as beta prime). Again, this structure can be cleavedby the DNAPs of the present invention to release a new alpha oligo.

[0220] At this point, the amplification has been linear. To increase thepower of the method, it is desired that the alpha signal oligohybridized to bead type 2 be liberated after release of the beta oligoso that it may go on to hybridize with other oligos on type 2 beads.Similarly, after release of an alpha oligo from type 3 beads, it isdesired that the beta oligo be liberated.

[0221] The liberation of “captured” signal oligos can be achieved in anumber of ways. First, it has been found that the DNAPs of the presentinvention have a true 5′ exonuclease capable of “nibbling” the 5′ end ofthe alpha (and beta) prime oligo (discussed below in more detail). Thus,under appropriate conditions, the hybridization is destabilized bynibbling of the DNAP. Second, the alpha - alpha prime (as well as thebeta—beta prime) complex can be destabilized by heat (e.g., thermalcycling).

[0222] With the liberation of signal oligos by such techniques, eachcleavage results in a doubling of the number of signal oligos. In thismanner, detectable signal can quickly be achieved.

[0223]FIG. 1B provides a schematic of a second embodiment of thedetection method of the present invention. Again, the target sequence isrecognized by two distinct oligonucleotides in the triggering or triggerreaction and the target nucleic acid aligns the two oligonucleotides forspecific cleavage of the 5′ arm by the DNAPs of the present invention(not shown in FIG. 1B). The first oligo is completely complementary to aportion of the target sequence. The second oligonucleotide is partiallycomplementary to the target sequence; the 3′ end of the secondoligonucleotide is fully complementary to the target sequence while the5′ end is non-complementary and forms a single-stranded arm. Thenon-complementary end of the second oligonucleotide may be a genericsequence which can be used with a set of standard hairpin structures(described below). The detection of different target sequences wouldrequire unique portions of two oligonucleotides: the entire firstoligonucleotide and the 3′ end of the second oligonucleotide. The 5′ armof the second oligonucleotide can be invariant or generic in sequence.

[0224] The annealing of the first and second oligonucleotides near oneanother along the target sequence forms a forked cleavage structurewhich is a substrate for the 5′ nuclease of DNA polymerases. Theapproximate location of the cleavage site is again indicated by thelarge solid arrowhead in FIG. 1B.

[0225] The 5′ nucleases of the invention are capable of cleaving thisstructure but are not capable of polymerizing the extension of the 3′end of the first oligonucleotide. The lack of polymerization activity isadvantageous as extension of the first oligonucleotide results indisplacement of the annealed region of the second oligonucleotide andresults in moving the site of cleavage along the second oligonucleotide.If polymerization is allowed to occur to any significant amount,multiple lengths of cleavage product will be generated. A singlecleavage product of uniform length is desirable as this cleavage productinitiates the detection reaction.

[0226] The trigger reaction may be run under conditions that allow forthermocycling. Thermocycling of the reaction allows for a logarithmicincrease in the amount of the trigger oligonucleotide released in thereaction.

[0227] The second part of the detection method allows the annealing ofthe fragment of the second oligonucleotide liberated by the cleavage ofthe first cleavage structure formed in the triggering reaction (calledthe third or trigger oligonucleotide) to a first hairpin structure. Thisfirst hairpin structure has a single-stranded 5′ arm and asingle-stranded 3′ arm. The third oligonucleotide triggers the cleavageof this first hairpin structure by annealing to the 3′ arm of thehairpin thereby forming a substrate for cleavage by the 5′ nuclease ofthe present invention. The cleavage of this first hairpin structuregenerates two reaction products: 1) the cleaved 5′ arm of the hairpincalled the fourth oligonucleotide, and 2) the cleaved hairpin structurewhich now lacks the 5′ arm and is smaller in size than the uncleavedhairpin. This cleaved first hairpin may be used as a detection moleculeto indicate that cleavage directed by the trigger or thirdoligonucleotide occurred. Thus, this indicates that the first twooligonucleotides found and annealed to the target sequence therebyindicating the presence of the target sequence in the sample.

[0228] The detection products are amplified by having the fourtholigonucleotide anneal to a second hairpin structure. This hairpinstructure has a 5′ single-stranded arm and a 3′ single-stranded arm. Thefourth oligonucleotide generated by cleavage of the first hairpinstructure anneals to the 3′ arm of the second hairpin structure therebycreating a third cleavage structure recognized by the 5′ nuclease. Thecleavage of this second hairpin structure also generates two reactionproducts: 1) the cleaved 5′ arm of the hairpin called the fiftholigonucleotide which is similar or identical in sequence to the thirdnucleotide, and 2) the cleaved second hairpin structure which now lacksthe 5′ arm and is smaller in size than the uncleaved hairpin. Thiscleaved second hairpin may be as a detection molecule and amplifies thesignal generated by the cleavage of the first hairpin structure.Simultaneously with the annealing of the forth oligonucleotide, thethird oligonucleotide is dissociated from the cleaved first hairpinmolecule so that it is free to anneal to a new copy of the first hairpinstructure. The disassociation of the oligonucleotides from the hairpinstructures may be accomplished by heating or other means suitable todisrupt base-pairing interactions.

[0229] Further amplification of the detection signal is achieved byannealing the fifth oligonucleotide (similar or identical in sequence tothe third oligonucleotide) to another molecule of the first hairpinstructure. Cleavage is then performed and the oligonucleotide that isliberated then is annealed to another molecule of the second hairpinstructure. Successive rounds of annealing and cleavage of the first andsecond hairpin structures, provided in excess, are performed to generatea sufficient amount of cleaved hairpin products to be detected. Thetemperature of the detection reaction is cycled just below and justabove the annealing temperature for the oligonucleotides used to directcleavage of the hairpin structures, generally about 55° C. to 70° C. Thenumber of cleavages will double in each cycle until the amount ofhairpin structures remaining is below the K_(m) for the hairpinstructures. This point is reached when the hairpin structures aresubstantially used up. When the detection reaction is to be used in aquantitative manner, the cycling reactions are stopped before theaccumulation of the cleaved hairpin detection products reach a plateau.

[0230] Detection of the cleaved hairpin structures may be achieved inseveral ways. In one embodiment detection is achieved by separation onagarose or polyacrylamide gels followed by staining with ethidiumbromide. In another embodiment, detection is achieved by separation ofthe cleaved and uncleaved hairpin structures on a gel followed byautoradiography when the hairpin structures are first labelled with aradioactive probe and separation on chromatography columns using HPLC orFPLC followed by detection of the differently sized fragments byabsorption at OD₂₆₀. Other means of detection include detection ofchanges in fluorescence polarization when the single-stranded 5′ arm isreleased by cleavage, the increase in fluorescence of an intercalatingfluorescent indicator as the amount of primers annealed to 3′ arms ofthe hairpin structures increases. The formation of increasing amounts ofduplex DNA (between the primer and the 3′ arm of the hairpin) occurs ifsuccessive rounds of cleavage occur.

[0231] The hairpin structures may be attached to a solid support, suchas an agarose, styrene or magnetic bead, via the 3′ end of the hairpin.A spacer molecule may be placed between the 3′ end of the hairpin andthe bead, if so desired. The advantage of attaching the hairpinstructures to a solid support is that this prevents the hybridization ofthe two hairpin structures to one another over regions which arecomplementary. If the hairpin structures anneal to one another, thiswould reduce the amount of hairpins available for hybridization to theprimers released during the cleavage reactions. If the hairpinstructures are attached to a solid support, then additional methods ofdetection of the products of the cleavage reaction may be employed.These methods include, but are not limited to, the measurement of thereleased single-stranded 5′ arm when the 5′ arm contains a label at the5′ terminus. This label may be radioactive, fluorescent, biotinylated,etc. If the hairpin structure is not cleaved, the S′ label will remainattached to the solid support. If cleavage occurs, the 5′ label will bereleased from the solid support.

[0232] The 3′ end of the hairpin molecule may be blocked through the useof dideoxynucleotides. A 3′ terminus containing a dideoxynucleotide isunavailable to participate in reactions with certain DNA modifyingenzymes, such as terminal transferase. Cleavage of the hairpin having a3′ terminal dideoxynucleotide generates a new, unblocked 3′ terminus atthe site of cleavage. This new 3′ end has a free hydroxyl group whichcan interact with terminal transferase thus providing another means ofdetecting the cleavage products.

[0233] The hairpin structures are designed so that theirself-complementary regions are very short (generally in the range of 3-8base pairs). Thus, the hairpin structures are not stable at the hightemperatures at which this reaction is performed (generally in the rangeof 50-75° C.) unless the hairpin is stabilized by the presence of theannealed oligonucleotide on the 3′ arm of the hairpin. This instabilityprevents the polymerase from cleaving the hairpin structure in theabsence of an associated primer thereby preventing false positiveresults due to non-oligonucleotide directed cleavage.

[0234] As discussed above, the use of the 5′ nucleases of the inventionwhich have reduced polymerization activity is advantageous in thismethod of detecting specific nucleic acid sequences. Significant amountsof polymerization during the cleavage reaction would cause shifting ofthe site of cleavage in unpredictable ways resulting in the productionof a series of cleaved hairpin structures of various sizes rather than asingle easily quantifiable product. Additionally, the primers used inone round of cleavage could, if elongated, become unusable for the nextcycle, by either forming an incorrect structure or by being too long tomelt off under moderate temperature cycling conditions. In a pristinesystem (i.e., lacking the presence of dNTPs), one could use theunmodified polymerase, but the presence of nucleotides (dNTPs) candecrease the per cycle efficiency enough to give a false negativeresult. When a crude extract (genomic DNA preparations, crude celllysates, etc.) is employed or where a sample of DNA from a PCR reaction,or any other sample that might be contaminated with dNTPs, the 5′nucleases of the present invention that were derived from thermostablepolymerases are particularly useful.

[0235] II. Generation of 5′ Nucleases from Thermostable DNA Polymerases

[0236] The genes encoding Type A DNA polymerases share about 85%homology to each other on the DNA sequence level. Preferred examples ofthermostable polymerases include those isolated from Thermus aquaticus,Thermus flavus, and Thermus thermophilus. However, other thermostableType A polymerases which have 5′ nuclease activity are also suitable.FIGS. 2 and 3 compare the nucleotide and amino acid sequences of thethree above mentioned polymerases. In FIGS. 2 and 3, the consensus ormajority sequence derived from a comparison of the nucleotide (FIG. 2)or amino acid (FIG. 3) sequence of the three thermostable DNApolymerases is shown on the top line. A dot appears in the sequences ofeach of these three polymerases whenever an amino acid residue in agiven sequence is identical to that contained in the consensus aminoacid sequence. Dashes are used to introduce gaps in order to maximizealignment between the displayed sequences. When no consensus nucleotideor amino acid is present at a given position, an “X” is placed in theconsensus sequence. SEQ ID NOS:1-3 display the nucleotide sequences andSEQ ID NOS:4-6 display the amino acid sequences of the three wild-typepolymerases. SEQ ID NO:1 corresponds to the nucleic acid sequence of thewild type Thermus aquaticus DNA polymerase gene isolated from the YT-1strain [Lawyer et al., J. Biol. Chem. 264:6427 (1989)]. SEQ ID NO:2corresponds to the nucleic acid sequence of the wild type Thermus flavusDNA polymerase gene [Akhmetzjanov and Vakhitov, Nucl. Acids Res. 20:5839(1992)]. SEQ ID NO:3 corresponds to the nucleic acid sequence of thewild type Thermus thermophilus DNA polymerase gene [Gelfand et al., WO91/09950 (1991)]. SEQ ID NOS:7-8 depict the consensus nucleotide andamino acid sequences, respectively for the above three DNAPs (also shownon the top row in FIGS. 2 and 3).

[0237] The 5′ nucleases of the invention derived from thermostablepolymerases have reduced synthetic ability, but retain substantially thesame 5′ exonuclease activity as the native DNA polymerase. The term“substantially the same 5′ nuclease activity” as used herein means thatthe 5′ nuclease activity of the modified enzyme retains the ability tofunction as a structure-dependent single-stranded endonuclease but notnecessarily at the same rate of cleavage as compared to the unmodifiedenzyme. Type A DNA polymerases may also be modified so as to produce anenzyme which has increases 5′ nuclease activity while having a reducedlevel of synthetic activity. Modified enzymes having reduced syntheticactivity and increased 5′ nuclease activity are also envisioned by thepresent invention.

[0238] By the term “reduced synthetic activity” as used herein it ismeant that the modified enzyme has less than the level of syntheticactivity found in the unmodified or “native” enzyme. The modified enzymemay have no synthetic activity remaining or may have that level ofsynthetic activity that will not interfere with the use of the modifiedenzyme in the detection assay described below. The 5′ nucleases of thepresent invention are advantageous in situations where the cleavageactivity of the polymerase is desired, but the synthetic ability is not(such as in the detection assay of the invention).

[0239] As noted above, it is not intended that the invention be limitedby the nature of the alteration necessary to render the polymerasesynthesis deficient. The present invention contemplates a variety ofmethods, including but not limited to: 1) proteolysis; 2) recombinantconstructs (including mutants); and 3) physical and/or chemicalmodification and/or inhibition.

[0240] 1. Proteolysis

[0241] Thermostable DNA polymerases having a reduced level of syntheticactivity are produced by physically cleaving the unmodified enzyme withproteolytic enzymes to produce fragments of the enzyme that aredeficient in synthetic activity but retain 5′ nuclease activity.Following proteolytic digestion, the resulting fragments are separatedby standard chromatographic techniques and assayed for the ability tosynthesize DNA and to act as a 5′ nuclease. The assays to determinesynthetic activity and 5′ nuclease activity are described below.

[0242] 2. Recombinant Constructs

[0243] The examples below describe a preferred method for creating aconstruct encoding a 5′ nuclease derived from a thermostable DNApolymerase. As the Type A DNA polymerases are similar in DNA sequence,the cloning strategies employed for the Thermus aquaticus and flavuspolymerases are applicable to other thermostable Type A polymerases. Ingeneral, a thermostable DNA polymerase is cloned by isolating genomicDNA using molecular biological methods from a bacteria containing athermostable Type A DNA polymerase. This genomic DNA is exposed toprimers which are capable of amplifying the polymerase gene by PCR.

[0244] This amplified polymerase sequence is then subjected to standarddeletion processes to delete the polymerase portion of the gene.Suitable deletion processes are described below in the examples.

[0245] The example below discusses the strategy used to determine whichportions of the DNAPTaq polymerase domain could be removed withouteliminating the 5′ nuclease activity. Deletion of amino acids from theprotein can be done either by deletion of the encoding genetic material,or by introduction of a translational stop codon by mutation or frameshift. In addition, proteolytic treatment of the protein molecule can beperformed to remove segments of the protein.

[0246] In the examples below, specific alterations of the Taq gene were:a deletion between nucleotides 1601 and 2502 (the end of the codingregion), a 4 nucleotide insertion at position 2043, and deletionsbetween nucleotides 1614 and 1848 and between nucleotides 875 and 1778(numbering is as in SEQ ID NO:1). These modified sequences are describedbelow in the examples and at SEQ ID NOS:9-12.

[0247] Those skilled in the art understand that single base pair changescan be innocuous in terms of enzyme structure and function. Similarly,small additions and deletions can be present without substantiallychanging the exonuclease or polymerase function of these enzymes.

[0248] Other deletions are also suitable to create the 5′ nucleases ofthe present invention. It is preferable that the deletion decrease thepolymerase activity of the 5′ nucleases to a level at which syntheticactivity will not interfere with the use of the 5′ nuclease in thedetection assay of the invention. Most preferably, the synthetic abilityis absent. Modified polymerases are tested for the presence of syntheticand 5′ nuclease activity as in assays described below. Thoughtfulconsideration of these assays allows for the screening of candidateenzymes whose structure is heretofore as yet unknown. In other words,construct “X” can be evaluated according to the protocol described belowto determine whether it is a member of the genus of 5′ nucleases of thepresent invention as defined functionally, rather than structurally.

[0249] In the example below, the PCR product of the amplified Thermusaquaticus genomic DNA did not have the identical nucleotide structure ofthe native genomic DNA and did not have the same synthetic ability ofthe original clone. Base pair changes which result due to the infidelityof DNAPTaq during PCR amplification of a polymerase gene are also amethod by which the synthetic ability of a polymerase gene may beinactivated. The examples below and FIGS. 4A and 5A indicate regions inthe native Thermus aquaticus and flavus DNA polymerases likely to beimportant for synthetic ability. There are other base pair changes andsubstitutions that will likely also inactivate the polymerase.

[0250] It is not necessary, however, that one start out the process ofproducing a 5′ nuclease from a DNA polymerase with such a mutatedamplified product. This is the method by which the examples below wereperformed to generate the synthesis-deficient DNAPTaq mutants, but it isunderstood by those skilled in the art that a wild-type DNA polymerasesequence may be used as the starting material for the introduction ofdeletions, insertion and substitutions to produce a 5′ nuclease. Forexample, to generate the synthesis-deficient DNAPTfl mutant, the primerslisted in SEQ ID NOS:13-14 were used to amplify the wild type DNApolymerase gene from Thermus flavus strain AT-62. The amplifiedpolymerase gene was then subjected to restriction enzyme digestion todelete a large portion of the domain encoding the synthetic activity.

[0251] The present invention contemplates that the nucleic acidconstruct of the present invention be capable of expression in asuitable host. Those in the art know methods for attaching variouspromoters and 3′ sequences to a gene structure to achieve efficientexpression. The examples below disclose two suitable vectors and sixsuitable vector constructs. Of course, there are other promoter/vectorcombinations that would be suitable. It is not necessary that a hostorganism be used for the expression of the nucleic acid constructs ofthe invention. For example, expression of the protein encoded by anucleic acid construct may be achieved through the use of a cell-free invitro transcription/translation system. An example of such a cell-freesystem is the commercially available TnT™ Coupled Reticulocyte LysateSystem (Promega Corporation, Madison, Wis.).

[0252] Once a suitable nucleic acid construct has been made, the 5′nuclease may be produced from the construct. The examples below andstandard molecular biological teachings enable one to manipulate theconstruct by different suitable methods.

[0253] Once the 5′ nuclease has been expressed, the polymerase is testedfor both synthetic and nuclease activity as described below.

[0254] 3. Physical and/or Chemical Modification and/or Inhibition

[0255] The synthetic activity of a thermostable DNA polymerase may bereduced by chemical and/or physical means. In one embodiment, thecleavage reaction catalyzed by the 5′ nuclease activity of thepolymerase is run under conditions which preferentially inhibit thesynthetic activity of the polymerase. The level of synthetic activityneed only be reduced to that level of activity which does not interferewith cleavage reactions requiring no significant synthetic activity.

[0256] As shown in the examples below, concentrations of Mg⁺⁺ greaterthan 5 mM inhibit the polymerization activity of the native DNAPTaq. Theability of the 5′ nuclease to function under conditions where syntheticactivity is inhibited is tested by running the assays for synthetic and5′ nuclease activity, described below, in the presence of a range ofMg⁺⁺ concentrations (5 to 10 mM). The effect of a given concentration ofMg⁺⁺ is determined by quantitation of the amount of synthesis andcleavage in the test reaction as compared to the standard reaction foreach assay.

[0257] The inhibitory effect of other ions, polyamines, denaturants,such as urea, formamide, dimethylsulfoxide, glycerol and non-ionicdetergents (Triton X-100 and Tween-20), nucleic acid binding chemicalssuch as, actinomycin D, ethidium bromide and psoralens, are tested bytheir addition to the standard reaction buffers for the synthesis and 5′nuclease assays. Those compounds having a preferential inhibitory effecton the synthetic activity of a thermostable polymerase are then used tocreate reaction conditions under which 5′ nuclease activity (cleavage)is retained while synthetic activity is reduced or eliminated.

[0258] Physical means may be used to preferentially inhibit thesynthetic activity of a polymerase. For example, the synthetic activityof thermostable polymerases is destroyed by exposure of the polymeraseto extreme heat (typically 96 to 100° C.) for extended periods of time(greater than or equal to 20 minutes). While these are minor differenceswith respect to the specific heat tolerance for each of the enzymes,these are readily determined. Polymerases are treated with heat forvarious periods of time and the effect of the heat treatment upon thesynthetic and 5′ nuclease activities is determined.

[0259] III. Detection of Specific Nucleic Acid Sequences Using 5′Nucleases in an Invader-Directed Cleavage Assay

[0260] The present invention provides means for forming a nucleic acidcleavage structure which is dependent upon the presence of a targetnucleic acid and cleaving the nucleic acid cleavage structure so as torelease distinctive cleavage products. 5′ nuclease activity is used tocleave the target-dependent cleavage structure and the resultingcleavage products are indicative of the presence of specific targetnucleic acid sequences in the sample.

[0261] The present invention further provides assays in which the targetnucleic acid is reused or recycled during multiple rounds ofhybridization with oligonucleotide probes and cleavage without the needto use temperature cycling (i.e., for periodic denaturation of targetnucleic acid strands) or nucleic acid synthesis (i.e., for thedisplacement of target nucleic acid strands). Through the interaction ofthe cleavage means (e.g., a 5′ nuclease) an upstream oligonucleotide,the cleavage means can be made to cleave a downstream oligonucleotide atan internal site in such a way that the resulting fragments of thedownstream oligonucleotide dissociate from the target nucleic acid,thereby making that region of the target nucleic acid available forhybridization to another, uncleaved copy of the downstreamoligonucleotide.

[0262] As illustrated in FIG. 29, the methods of the present inventionemploy at least a pair of oligonucleotides that interact with a targetnucleic acid to form a cleavage structure for a structure-specificnuclease. More specifically, the cleavage structure comprises i) atarget nucleic acid that may be either single-stranded ordouble-stranded (when a double-stranded target nucleic acid is employed,it may be rendered single stranded, e.g., by heating); ii) a firstoligonucleotide, termed the “probe,” which defines a first region of thetarget nucleic acid sequence by being the complement of that region(regions X and Z of the target as shown in FIG. 29); iii) a secondoligonucleotide, termed the “invader,” the 5′ part of which defines asecond region of the same target nucleic acid sequence (regions Y and Xin FIG. 29), adjacent to and downstream of the first target region(regions X and Z), and the second part of which overlaps into the regiondefined by the first oligonucleotide (region X depicts the region ofoverlap). The resulting structure is diagrammed in FIG. 29.

[0263] While not limiting the invention or the instant discussion to anyparticular mechanism of action, the diagram in FIG. 29 represents theeffect on the site of cleavage caused by this type of arrangement of apair of oligonucleotides. The design of such a pair of oligonucleotidesis described below in detail. In FIG. 29, the 3′ ends of the nucleicacids (i.e., the target and the oligonucleotides) are indicated by theuse of the arrowheads on the ends of the lines depicting the strands ofthe nucleic acids (and where space permits, these ends are also labelled“3′”). It is readily appreciated that the two oligonucleotides (theinvader and the probe) are arranged in a parallel orientation relativeto one another, while the target nucleic acid strand is arranged in ananti-parallel orientation relative to the two oligonucleotides. Furtherit is clear that the invader oligonucleotide is located upstream of theprobe oligonucleotide and that with respect to the target nucleic acidstrand, region Z is upstream of region X and region X is upstream ofregion Y (that is region Y is downstream of region X and region X isdownstream of region Z). Regions of complementarity between the opposingstrands are indicated by the short vertical lines. While not intended toindicate the precise location of the site(s) of cleavage, the area towhich the site of cleavage within the probe oligonucleotide is shiftedby the presence of the invader oligonucleotide is indicated by the solidvertical arrowhead. An alternative representation of thetarget/invader/probe cleavage structure is shown in FIG. 32c. Neitherdiagram (i.e., FIG. 29 or FIG. 32c) is intended to represent the actualmechanism of action or physical arrangement of the cleavage structureand further it is not intended that the method of the present inventionbe limited to any particular mechanism of action.

[0264] It can be considered that the binding of these oligonucleotidesdivides the target nucleic acid into three distinct regions: one regionthat has complementarity to only the probe (shown as “Z”); one regionthat has complementarity only to the invader (shown as “Y”); and oneregion that has complementarity to both oligonucleotides (shown as “X”).

[0265] Design of these oligonucleotides (i.e., the invader and theprobe) is accomplished using practices which are standard in the art.For example, sequences that have self complementarity, such that theresulting oligonucleotides would either fold upon themselves, orhybridize to each other at the expense of binding to the target nucleicacid, are generally avoided.

[0266] One consideration in choosing a length for these oligonucleotidesis the complexity of the sample containing the target nucleic acid. Forexample, the human genome is approximately 3×10⁹ basepairs in length.Any 10 nucleotide sequence will appear with a frequency of 1:4¹⁰, or1:1048,576 in a random string of nucleotides, which would beapproximately 2,861 times in 3 billion basepairs. Clearly anoligonucleotide of this length would have a poor chance of bindinguniquely to a 10 nucleotide region within a target having a sequence thesize of the human genome. If the target sequence were within a 3 kbplasmid, however, such an oligonucleotide might have a very reasonablechance of binding uniquely. By this same calculation it can be seen thatan oligonucleotide of 16 nucleotides (i.e., a 16-mer) is the minimumlength of a sequence which is mathematically likely to appear once in3×10⁹ basepairs.

[0267] A second consideration in choosing oligonucleotide length is thetemperature range in which the oligonucleotides will be expected tofunction. A 16-mer of average base content (50% G-C basepairs) will havea calculated T_(m) (the temperature at which 50% of the sequence isdissociated) of about 41° C., depending on, among other things, theconcentration of the oligonucleotide and its target, the salt content ofthe reaction and the precise order of the nucleotides. As a practicalmatter, longer oligonucleotides are usually chosen to enhance thespecificity of hybridization. Oligonucleotides 20 to 25 nucleotides inlength are often used as they are highly likely to be specific if usedin reactions conducted at temperatures which are near their T_(m)s(within about 5° of the T_(m)). In addition, with calculated T_(m)s inthe range of 50° to 70° C., such oligonucleotides (i.e, 20 to 25-mers)are appropriately used in reactions catalyzed by thermostable enzymes,which often display optimal activity near this temperature range.

[0268] The maximum length of the oligonucleotide chosen is also based onthe desired specificity. One must avoid choosing sequences that are solong that they are either at a high risk of binding stably to partialcomplements, or that they cannot easily be dislodged when desired (e.g.,failure to disassociate from the target once cleavage has occurred).

[0269] The first step of design and selection of the oligonucleotidesfor the invader-directed cleavage is in accordance with these samplegeneral principles. Considered as sequence-specific probes individually,each oligonucleotide may be selected according to the guidelines listedabove. That is to say, each oligonucleotide will generally be longenough to be reasonably expected to hybridize only to the intendedtarget sequence within a complex sample, usually in the 20 to 40nucleotide range. Alternatively, because the invader-directed cleavageassay depends upon the concerted action of these oligonucleotides, thecomposite length of the 2 oligonucleotides which span/bind to the X, Y,Z regions may be selected to fall within this range, with each of theindividual oligonucleotides being in approximately the 13 to 17nucleotide range. Such a design might be employed if a non-thermostablecleavage means were employed in the reaction, requiring the reactions tobe conducted at a lower temperature than that used when thermostablecleavage means are employed. In some instances, it may be desirable tohave these oligonucleotides bind multiple times within a target nucleicacid (e.g., which bind to multiple variants or multiple similarsequences within a target). It is not intended that the method of thepresent invention be limited to any particular size of the probe orinvader oligonucleotide.

[0270] The second step of designing an oligonucleotide pair for thisassay is to choose the degree to which the upstream “invader”oligonucleotide sequence will overlap into the downstream “probe”oligonucleotide sequence, and consequently, the sizes into which theprobe will be cleaved. A key feature of this assay is that the probeoligonucleotide can be made to “turn over,” that is to say cleaved probecan be made to depart to allow the binding and cleavage of other copiesof the probe molecule, without the requirements of thermal denaturationor displacement by polymerization. While in one embodiment of this assayprobe turnover may be facilitated by an exonucleolytic digestion by thecleavage agent, it is central to the present invention that the turnoverdoes not require this exonucleolytic activity.

[0271] Choosing the Amount of Overlap (Length of the X Region)

[0272] One way of accomplishing such turnover can be envisioned byconsidering the diagram in FIG. 29. It can be seen that the Tm of eacholigonucleotide will be a function of the full length of thatoligonucleotide: i.e., the T_(m) of the invader=Tm(Y+X), and the Tm ofthe probe=Tm_((X+Y)) for the probe. When the probe is cleaved the Xregion is released, leaving the Z section. If the Tm of Z is less thanthe reaction temperature, and the reaction temperature is less than theTm_((X+Z)), then cleavage of the probe will lead to the departure of Z,thus allowing a new (X+Z) to hybridize. It can be seen from this examplethat the X region must be sufficiently long that the release of X willdrop the Tm of the remaining probe section below the reactiontemperature: a G-C rich X section may be much shorter than an A-T rich Xsection and still accomplish this stability shift.

[0273] Designing Oligonucleotides which Interact with the Y and ZRegions

[0274] If the binding of the invader oligonucleotide to the target ismore stable than the binding of the probe (e.g., if it is long, or isrich in G-C basepairs in the Y region), then the copy of X associatedwith the invader may be favored in the competition for binding to the Xregion of the target, and the probe may consequently hybridizeinefficiently, and the assay may give low signal. Alternatively, if theprobe binding is particularly strong in the Z region, the invader willstill cause internal cleavage, because this is mediated by the enzyme,but portion of the probe oligonucleotide bound to the Z region may notdissociate at the reaction temperature, turnover may be poor, and theassay may again give low signal.

[0275] It is clearly beneficial for the portions of the oligonucleotidewhich interact with the Y and Z regions so be similar in stability,i.e., they must have similar melting temperatures. This is not to saythat these regions must be the same length. As noted above, in additionto length, the melting temperature will also be affected by the basecontent and the specific sequence of those bases. The specific stabilitydesigned into the invader and probe sequences will depend on thetemperature at which one desires to perform the reaction.

[0276] This discussion is intended to illustrate that (within the basicguidelines for oligonucleotide specificity discussed above) it is thebalance achieved between the stabilities of the probe and invadersequences and their X and Y component sequences, rather than theabsolute values of these stabilities, that is the chief consideration inthe selection of the probe and invader sequences.

[0277] Design of the Reaction Conditions

[0278] Target nucleic acids that may be analyzed using the methods ofthe present invention which employ a 5′ nuclease as the cleavage meansinclude many types of both RNA and DNA. Such nucleic acids may beobtained using standard molecular biological techniques. For example,nucleic acids (RNA or DNA) may be isolated from a tissue sample (e.g, abiopsy specimen), tissue culture cells, samples containing bacteriaand/or viruses (including cultures of bacteria and/or viruses), etc. Thetarget nucleic acid may also be transcribed in vitro from a DNA templateor may be chemically synthesized or generated in a PCR. Furthermore,nucleic acids may be isolated from an organism, either as genomicmaterial or as a plasmid or similar extrachromosomal DNA, or they may bea fragment of such material generated by treatment with a restrictionendonuclease or other cleavage agents or it may be synthetic.

[0279] Assembly of the target, probe, and invader nucleic acids into thecleavage reaction of the present invention uses principles commonly usedin the design of oligonucleotide base enzymatic assays, such asdideoxynucleotide sequencing and polymerase chain reaction (PCR). As isdone in these assays, the oligonucleotides are provided in sufficientexcess that the rate of hybridization to the target nucleic acid is veryrapid. These assays are commonly performed with 50 fmoles to 2 pmoles ofeach oligonucleotide per μl of reaction mixture. In the Examplesdescribed herein, amounts of oligonucleotides ranging from 250 fmoles to5 pmoles per μl of reaction volume were used. These values were chosenfor the purpose of ease in demonstration and are not intended to limitthe performance of the present invention to these concentrations. Other(e.g., lower) oligonucleotide concentrations commonly used in othermolecular biological reactions are also contemplated.

[0280] It is desirable that an invader oligonucleotide be immediatelyavailable to direct the cleavage of each probe oligonucleotide thathybridizes to a target nucleic acid. For this reason, in the Examplesdescribed herein, the invader oligonucleotide is provided in excess overthe probe oligonucleotide; often this excess is 10-fold. While this isan effective ratio, it is not intended that the practice of the presentinvention be limited to any particular ratio of invader-to-probe (aratio of 2- to 100-fold is contemplated).

[0281] Buffer conditions must be chosen that will be compatible withboth the oligonucleotide/target hybridization and with the activity ofthe cleavage agent. The optimal buffer conditions for nucleic acidmodification enzymes, and particularly DNA modification enzymes,generally included enough mono- and di-valent salts to allow associationof nucleic acid strands by base-pairing. If the method of the presentinvention is performed using an enzymatic cleavage agent other thanthose specifically described here, the reactions may generally beperformed in any such buffer reported to be optimal for the nucleasefunction of the cleavage agent. In general, to test the utility of anycleavage agent in this method, test reactions are performed wherein thecleavage agent of interest is tested in the MOPS/MnCl₂/KCl buffer orMg-containing buffers described herein and in whatever buffer has beenreported to be suitable for use with that agent, in a manufacturer'sdata sheet, a journal article, or in personal communication.

[0282] The products of the invader-directed cleavage reaction arefragments generated by structure-specific cleavage of the inputoligonucleotides. The resulting cleaved and/or uncleavedoligonucleotides may be analyzed and resolved by a number of methodsincluding electrophoresis (on a variety of supports including acrylamideor agarose gels, paper, etc.), chromatography, fluorescencepolarization, mass spectrometry and chip hybridization. The invention isillustrated using electrophoretic separation for the analysis of theproducts of the cleavage reactions. However, it is noted that theresolution of the cleavage products is not limited to electrophoresis.Electrophoresis is chosen to illustrate the method of the inventionbecause electrophoresis is widely practiced in the art and is easilyaccessible to the average practioner.

[0283] The probe and invader oligonucleotides may contain a label to aidin their detection following the cleavage reaction. The label may be aradioisotope (e.g., a ³²P or ³S-labelled nucleotide) placed at eitherthe 5′ or 3′ end of the oligonucleotide or alternatively, the label maybe distributed throughout the oligonucleotide (i.e., a uniformlylabelled oligonucleotide). The label may be a nonisotopic detectablemoiety, such as a fluorophore, which can be detected directly, or areactive group which permits specific recognition by a secondary agent.For example, biotinylated oligonucleotides may be detected by probingwith a streptavidin molecule which is coupled to an indicator (e.g.,alkaline phosphatase or a fluorophore) or a hapten such as dioxigeninmay be detected using a specific antibody coupled to a similarindicator.

[0284] Optimization of Reaction Conditions

[0285] The invader-directed cleavage reaction is useful to detect thepresence of specific nucleic acids. In addition to the considerationslisted above for the selection and design of the invader and probeoligonucleotides, the conditions under which the reaction is to beperformed may be optimized for detection of a specific target sequence.

[0286] One objective in optimizing the invader-directed cleavage assayis to allow specific detection of the fewest copies of a target nucleicacid. To achieve this end, it is desirable that the combined elements ofthe reaction interact with the maximum efficiency, so that the rate ofthe reaction (e.g., the number of cleavage events per minute) ismaximized. Elements contributing to the overall efficiency of thereaction include the rate of hybridization, the rate of cleavage, andthe efficiency of the release of the cleaved probe.

[0287] The rate of cleavage will be a function of the cleavage meanschosen, and may be made optimal according to the manufacturer'sinstructions when using commercial preparations of enzymes or asdescribed in the examples herein. The other elements (rate ofhybridization, efficiency of release) depend upon the execution of thereaction, and optimization of these elements is discussed below.

[0288] Three elements of the cleavage reaction that significantly affectthe rate of nucleic acid hybridization are the concentration of thenucleic acids, the temperature at which the cleavage reaction isperformed and the concentration of salts and/or other charge-shieldingions in the reaction solution.

[0289] The concentrations at which oligonucleotide probes are used inassays of this type are well known in the art, and are discussed above.One example of a common approach to optimizing an oligonucleotideconcentration is to choose a starting amount of oligonucleotide forpilot tests; 0.01 to 2 μM is a concentration range used in manyoligonucleotide-based assays. When initial cleavage reactions areperformed, the following questions may be asked of the data: Is thereaction performed in the absence of the target nucleic acidsubstantially free of the cleavage product?; Is the site of cleavagespecifically shifted in accordance with the design of the invaderoligonucleotide?; Is the specific cleavage product easily detected inthe presence of the uncleaved probe (or is the amount of uncut materialoverwhelming the chosen visualization method)?

[0290] A negative answer to any of these questions would suggest thatthe probe concentration is too high, and that a set of reactions usingserial dilutions of the probe should be performed until the appropriateamount is identified. Once identified for a given target nucleic acid ina give sample type (e.g., purified genomic DNA, body fluid extract,lysed bacterial extract), it should not need to be re-optimized. Thesample type is important because the complexity of the material presentmay influence the probe optimum.

[0291] Conversely, if the chosen initial probe concentration is too low,the reaction may be slow, due to inefficient hybridization. Tests withincreasing quantities of the probe will identify the point at which theconcentration exceeds the optimum. Since the hybridization will befacilitated by excess of probe, it is desirable, but not required, thatthe reaction be performed using probe concentrations just below thispoint.

[0292] The concentration of invader oligonucleotide can be chosen basedon the design considerations discussed above. In a preferred embodiment,the invader oligonucleotide is in excess of the probe oligonucleotide.In a particularly preferred embodiment, the invader is approximately10-fold more abundant than the probe.

[0293] Temperature is also an important factor in the hybridization ofoligonucleotides. The range of temperature tested will depend in largepart, on the design of the oligonucleotides, as discussed above. In apreferred embodiment, the reactions are performed at temperaturesslightly below the Tm of the least stable oligonucleotide in thereaction. Melting temperatures for the oligonucleotides and for theircomponent regions (X, Y and Z, FIG. 29), can be estimated through theuse of computer software or, for a more rough approximation, byassigning the value of 2° C. per A-T basepair, and 4° C. per G-Cbasepair, and taking the sum across an expanse of nucleic acid. Thelatter method may be used for oligonucleotides of approximately 10-30nucleotides in length. Because even computer prediction of the Tm of anucleic acid is only an approximation, the reaction temperatures chosenfor initial tests should bracket the calculated T_(m). Whileoptimizations are not limited to this, 5° C. increments are convenienttest intervals in these optimization assays.

[0294] When temperatures are tested, the results can be analyzed forspecificity (the first two of the questions listed above) in the sameway as for the oligonucleotide concentration determinations.Non-specific cleavage (i.e., cleavage of the probe at many or allpositions along its length) would indicate non-specific interactionsbetween the probe and the sample material, and would suggest that ahigher temperature should be employed. Conversely, little or no cleavagewould suggest that even the intended hybridization is being prevented,and would suggest the use of lower temperatures. By testing severaltemperatures, it is possible to identify an approximate temperatureoptimum, at which the rate of specific cleavage of the probe is highest.If the oligonucleotides have been designed as described above, the Tm ofthe Z-region of the probe oligonucleotide should be below thistemperature, so that turnover is assured.

[0295] A third determinant of hybridization efficiency is the saltconcentration of the reaction. In large part, the choice of solutionconditions will depend on the requirements of the cleavage agent, andfor reagents obtained commercially, the manufacturer's instructions area resource for this information. When developing an assay utilizing anyparticular cleavage agent, the oligonucleotide and temperatureoptimizations described above should be performed in the bufferconditions best suited to that cleavage agent.

[0296] A “no enzyme” control allows the assessment of the stability ofthe labeled oligonucleotides under particular reaction conditions, or inthe presence of the sample to be tested (i.e., in assessing the samplefor contaminating nucleases). In this manner, the substrate andoligonucleotides are placed in a tube containing all reactioncomponents, except the enzyme and treated the same as theenzyme-containing reactions. Other controls may also be included. Forexample, a reaction with all of the components except the target nucleicacid will serve to confirm the dependence of the cleavage on thepresence of the target sequence.

[0297] Probing for Multiple Alleles

[0298] The invader-directed cleavage reaction is also useful in thedetection and quantification of individual variants or alleles in amixed sample population. By way of example, such a need exists in theanalysis of tumor material for mutations in genes associated withcancers. Biopsy material from a tumor can have a significant complementof normal cells, so it is desirable to detect mutations even whenpresent in fewer than 5% of the copies of the target nucleic acid in asample. In this case, it is also desirable to measure what fraction ofthe population carries the mutation. Similar analyses may also be doneto examine allelic variation in other gene systems, and it is notintended that the method of the present invention by limited to theanalysis of tumors.

[0299] As demonstrated below, reactions can be performed underconditions that prevent the cleavage of probes bearing even asingle-nucleotide difference mismatch within the region of the targetnucleic acid termed “Z” in FIG. 29, but that permit cleavage of asimilar probe that is completely complementary to the target in thisregion. Thus, the assay may be used to quantitate individual variants oralleles within a mixed sample.

[0300] The use of multiple, differently labelled probes in such an assayis also contemplated. To assess the representation of different variantsor alleles in a sample, one would provide a mixture of probes such thateach allele or variant to be detected would have a specific probe (i.e.,perfectly matched to the Z region of the target sequence) with a uniquelabel (e.g., no two variant probes with the same label would be used ina single reaction). These probes would be characterized in advance toensure that under a single set of reaction conditions, they could bemade to give the same rate of signal accumulation when mixed with theirrespective target nucleic acids. Assembly of a cleavage reactioncomprising the mixed probe set, a corresponding invader oligonucleotide,the target nucleic acid sample, and the appropriate cleavage agent,along with performance of the cleavage reaction under conditions suchthat only the matched probes would cleave, would allow independentquantification of each of the species present, and would thereforeindicate their relative representation in the target sample.

[0301] IV. A Comparision of Invasive Cleavage and Primer-DirectedCleavage

[0302] As discussed herein, the terms “invasive” or “invader-directed”cleavage specifically denote the use of a first, upstreamoligonucleotide, as defined below, to cause specific cleavage at a sitewithin a second, downstream sequence. To effect such a direction ofcleavage to a region within a duplex, it is required that the first andsecond oligonucleotides overlap in sequence. That is to say, a portionof the upstream oligonucleotide, termed the “invader”, has significanthomology to a portion of the downstream “probe” oligonucleotide, so thatthese regions would tend to basepair with the same complementary regionof the target nucleic acid to be detected. While not limiting thepresent invention to any particular mechanism, the overlapping regionswould be expected to alternate in their occupation of the sharedhybridization site. When the probe oligonucleotide fully anneals to thetarget nucleic acid, and thus forces the 3′ region of the invader toremain unpaired, the structure so formed is not a substrate for the 5′nucleases of the present invention. By contrast, when the inverse istrue, the structure so formed is substrate for these enzymes, allowingcleavage and release of the portion of the probe oligonucleotide that isdisplaced by the invader oligonucleotide. The shifting of the cleavagesite to a region the probe oligonucleotide that would otherwise bebasepaired to the target sequence is one hallmark of the invasivecleavage assay (i.e., the invader-directed cleavage assay) of thepresent invention.

[0303] It is beneficial at this point to contrast the invasive cleavageas described above with two other forms of probe cleavage that may leadto internal cleavage of a probe oligonucleotide, but which do notcomprise invasive cleavage. In the first case, a hybridized probe may besubject to duplex-dependent 5′ to 3′ exonuclease “nibbling,” such thatthe oligonucleotide is shortened from the 5′ end until it cannot remainbound to the target (see, e.g., Examples 6-8 and FIGS. 26-28). The siteat which such nibbling stops can appear to be discrete, and, dependingon the difference between the melting temperature of the full-lengthprobe and the temperature of the reaction, this stopping point may be 1or several nucleotides into the probe oligonucleotide sequence. Such“nibbling” is often indicated by the presence of a “ladder” of longerproducts ascending size up to that of the full length of the probe, butthis is not always the case. While any one of the products of such anibbling reaction may be made to match in size and cleavage site theproducts of an invasive cleavage reaction, the creation of thesenibbling products would be highly dependent on the temperature of thereaction and the nature of the cleavage agent, but would be independentof the action of an upstream oligonucleotide, and thus could not beconstrued to involve invasive cleavage.

[0304] A second cleavage structure that may be considered is one inwhich a probe oligonucleotide has several regions of complementaritywith the target nucleic acid, interspersed with one or more regions ornucleotides of noncomplementarity. These noncomplementary regions may bethought of as “bubbles” within the nucleic acid duplex. As temperatureis elevated, the regions of complementarity can be expected to “melt” inthe order of their stability, lowest to highest. When a region of lowerstability is near the end of a segment of duplex, and the next region ofcomplementarity along the strand has a higher melting temperature, atemperature can be found that will cause the terminal region of duplexto melt first, opening the first bubble, and thereby creating apreferred substrate structure of the cleavage by the 5′ nucleases of thepresent invention (FIG. 40a). The site of such cleavage would beexpected to be on the 5′ arm, within 2 nucleotides of the junctionbetween the single and double-stranded regions (Lyamichev et al., supraand U.S. Pat. No. 5,422,253).

[0305] An additional oligonucleotide could be introduced to basepairalong the target nucleic acid would have a similar effect of openingthis bubble for subsequent cleavage of the unpaired 5′ arm (FIG. 40b andFIG. 6). Note in this case, the 3′ terminal nucleotides of the upstreamoligonucleotide anneals along the target nucleic acid sequence in such amanner that the 3′ end is located within the “bubble” region. Dependingon the precise location of the 3′ end of this oligonucleotide, thecleavage site may be along the newly unpaired 5′ arm, or at the siteexpected for the thermally opened bubble structure as described above.In the former case the cleavage is not within a duplexed region, and isthus not invasive cleavage, while in the latter the oligonucleotide ismerely an aide in inducing cleavage at a site that might otherwise beexposed through the use of temperature alone (i.e., in the absence ofthe additional oligonucleotide), and is thus not considered to beinvasive cleavage.

[0306] In summary, any arrangement of oligonucleotides used for thecleavage-based detection of a target sequence can be analyzed todetermine if the arrangement is an invasive cleavage structure ascontemplated herein. An invasive cleavage structure supports cleavage ofthe probe in a region that, in the absence of an upstreamoligonucleotide, would be expected to be basepaired to the targetnucleic acid.

[0307] Example 26 below provides further guidance for the design andexecution of a experiments which allow the determination of whether agiven arrangement of a pair of upstream and downstream (i.e., the probe)oligonucleotides when annealed along a target nucleic acid would form aninvasive cleavage structure.

[0308] V. Fractionation of Specific Nucleic Acids by Selective ChargeReversal

[0309] Some nucleic acid-based detection assays involve the elongationand/or shortening of oligonucleotide probes. For example, as describedherein, the primer-directed, primer-independent, and invader-directedcleavage assays, as well as the “nibbling” assay all involve thecleavage (i.e., shortening) of oligonucleotides as a means for detectingthe presence of a target nucleic sequence. Examples of other detectionassays which involve the shortening of an oligonucleotide probe includethe “TaqMan” or nick-translation PCR assay described in U.S. Pat. No.5,210,015 to Gelfand et al. (the disclosure of which is hereinincorporated by reference), the assays described in U.S. Pat. Nos.4,775,619 and 5,118,605 to Urdea (the disclosures of which are hereinincorporated by reference), the catalytic hybridization amplificationassay described in U.S. Pat. No. 5,403,711 to Walder and Walder (thedisclosure of which is herein incorporated by reference), and thecycling probe assay described in U.S. Pat. Nos. 4,876,187 and 5,011,769to Duck et al. (the disclosures of which are herein incorporated byreference). Examples of detection assays which involve the elongation ofan oligonucleotide probe (or primer) include the polymerase chainreaction (PCR) described in U.S. Pat. Nos. 4,683,195 and 4,683,202 toMullis and Mullis et al. (the disclosures of which are hereinincorporated by reference) and the ligase chain reaction (LCR) describedin U.S. Pat. Nos. 5,427,930 and 5,494,810 to Birkenmeyer et al. andBarany et al. (the disclosures of which are herein incorporated byreference). The above examples are intended to be illustrative ofnucleic acid-based detection assays that involve the elongation and/orshortening of oligonucleotide probes and do not provide an exhaustivelist.

[0310] Typically, nucleic acid-based detection assays that involve theelongation and/or shortening of oligonucleotide probes requirepost-reaction analysis to detect the products of the reaction. It iscommon that, the specific reaction product(s) must be separated from theother reaction components, including the input or unreactedoligonucleotide probe. One detection technique involves theelectrophoretic separation of the reacted and unreacted oligonucleotideprobe. When the assay involves the cleavage or shortening of the probe,the unreacted product will be longer than the reacted or cleavedproduct. When the assay involves the elongation of the probe (orprimer), the reaction products will be greater in length than the input.Gel-based electrophoresis of a sample containing nucleic acid moleculesof different lengths separates these fragments primarily on the basis ofsize. This is due to the fact that in solutions having a neutral oralkaline pH, nucleic acids having widely different sizes (i.e.,molecular weights) possess very similar charge-to-mass ratios and do notseparate [Andrews, Electrophoresis, 2nd Edition, Oxford University Press(1986), pp. 153-154]. The gel matrix acts as a molecular sieve andallows nucleic acids to be separated on the basis of size and shape(e.g., linear, relaxed circular or covalently closed supercoiledcircles).

[0311] Unmodified nucleic acids have a net negative charge due to thepresence of negatively charged phosphate groups contained within thesugar-phosphate backbone of the nucleic acid. Typically, the sample isapplied to gel near the negative pole and the nucleic acid fragmentsmigrate into the gel toward the positive pole with the smallestfragments moving fastest through the gel.

[0312] The present invention provides a novel means for fractionatingnucleic acid fragments on the basis of charge. This novel separationtechnique is related to the observation that positively charged adductscan affect the electrophoretic behavior of small oligonucleotidesbecause the charge of the adduct is significant relative to charge ofthe whole complex. In addition, to the use of positively charged adducts(e.g., Cy3 and CyS amidite fluorescent dyes, the positively chargedheterodimeric DNA-binding dyes shown in FIG. 66, etc.), theoligonucleotide may contain amino acids (particulary useful amino acidsare the charged amino acids: lysine, arginine, asparate, glutamate),modified bases, such as amino-modified bases, and/or a phosphonatebackbone (at all or a subset of the positions). In addition as discussedfurther below, a neutral dye or detection moiety (e.g., biotin,streptavidin, etc.) may be employed in place of a positively chargedadduct in conjunction with the use of amino-modified bases and/or acomplete or partial phosphonate backbone.

[0313] This observed effect is of particular utility in assays based onthe cleavage of DNA molecules. Using the assays described herein as anexample, when an oligonucleotide is shortened through the action of aCleavase® enzyme or other cleavage agent, the positive charge can bemade to not only significantly reduce the net negative charge, but toactually override it, effectively “flipping” the net charge of thelabeled entity. This reversal of charge allows the products oftarget-specific cleavage to be partitioned from uncleaved probe byextremely simple means. For example, the products of cleavage can bemade to migrate towards a negative electrode placed at any point in areaction vessel, for focused detection without gel-basedelectrophoresis; Example 24 provides examples of devices suitable forfocused detection without gel-based electrophoresis. When a slab gel isused, sample wells can be positioned in the center of the gel, so thatthe cleaved and uncleaved probes can be observed to migrate in oppositedirections. Alternatively, a traditional vertical gel can be used, butwith the electrodes reversed relative to usual DNA gels (i.e., thepositive electrode at the top and the negative electrode at the bottom)so that the cleaved molecules enter the gel, while the uncleaveddisperse into the upper reservoir of electrophoresis buffer.

[0314] An important benefit of this type of readout is the absolutenature of the partition of products from substrates, i.e., theseparation is virtually 100%. This means that an abundance of uncleavedprobe can be supplied to drive the hybridization step of the probe-basedassay, yet the unconsumed (i.e., unreacted) probe can, in essence, besubtracted from the result to reduce background by virtue of the factthat the unreacted probe will not migrate to the same pole as thespecific reaction product.

[0315] Through the use of multiple positively charged adducts, syntheticmolecules can be constructed with sufficient modification that thenormally negatively charged strand is made nearly neutral. When soconstructed, the presence or absence of a single phosphate group canmean the difference between a net negative or a net positive charge.This observation has particular utility when one objective is todiscriminate between enzymatically generated fragments of DNA, whichlack a 3′ phosphate, and the products of thermal degradation, whichretain a 3′ phosphate (and thus two additional negative charges).Examples 23 and 24 demonstrate the ability to separate positivelycharged reaction products from a net negatively charged substrateoligonucleotide. As discussed in these examples, oligonucleotides may betransformed from net negative to net positively charged compounds. InExample 24, the positively charged dye, Cy3 was incorporated at the 5′end of a 22-mer (SEQ ID NO:61) which also contained twoamino-substituted residues at the 5′ end of the oligonucleotide; thisoligonucleotide probe carries a net negative charge. After cleavage,which occurred 2 nucleotides into the probe, the following labelledoligonucleotide was released: 5′-Cy3-AminoT-AminoT-3′ (as well as theremaining 20 nucleotides of SEQ ID NO:61). This short fragment bears anet positive charge while the reaminder of the cleaved oligonucleotideand the unreacted or input oligonucleotide bear net negative charges.

[0316] The present invention contemplates embodiments wherein thespecific reaction product produced by any cleavage of anyoligonucleotide can be designed to carry a net positive charge while theunreacted probe is charge neutral or carries a net negative charge. Thepresent invention also contemplates embodiments where the releasedproduct may be designed to carry a net negative charge while the inputnucleic acid carries a net positive charge. Depending on the length ofthe released product to be detected, positively charged dyes may beincorporated at the one end of the probe and modified bases may beplaced along the oligonucleotide such that upon cleavage, the releasedfragment containing the positively charged dye carries a net positivecharge. Amino-modified bases may be used to balance the charge of thereleased fragment in cases where the presence of the positively chargedadduct (e.g., dye) alone is not sufficient to impart a net positivecharge on the released fragment. In addition, the phosphate backbone maybe replaced with a phosphonate backbone at a level sufficient to imparta net positive charge (this is particularly useful when the sequence ofthe oligonucleotide is not amenable to the use of amino-substitutedbases); FIGS. 56 and 57 show the structure of short oligonucleotidescontaining a phosphonate group on the second T residue). Anoligonucleotide containing a fully phosphonate-substituted backbonewould be charge neutral (absent the presence of modified chargedresidues bearing a charge or the presence of a charged adduct) due tothe absence of the negatively charged phosphate groups.Phosphonate-containing nucleotides (e.g., methylphosphonate-containingnucleotides are readily available and can be incorporated at anyposition of an oligonucleotide during synthesis using techniques whichare well known in the art.

[0317] In essence, the invention contemplates the use of charge-basedseparation to permit the separation of specific reaction products fromthe input oligonucleotides in nucleic acid-based detection assays. Thefoundation of this novel separation technique is the design and use ofoligonucleotide probes (typically termed “primers” in the case of PCR)which are “charge balanced” so that upon either cleavage or elongationof the probe it becomes “charge unbalanced,” and the specific reactionproducts may be separated from the input reactants on the basis of thenet charge.

[0318] In the context of assays which involve the elongation of anoligonucleotide probe (i.e., a primer), such as is the case in PCR, theinput primers are designed to carry a net positive charge. Elongation ofthe short oligonucleotide primer during polymerization will generate PCRproducts which now carry a net negative charge. The specific reactionproducts may then easily be separated and concentrated away from theinput primers using the charge-based separation technique describedherein (the electrodes will be reversed relative to the description inExample 24 as the product to be separated and concentrated after a PCRwill carry a negative charge).

Experimental

[0319] The following examples serve to illustrate certain preferredembodiments and aspects of the present invention and are not to beconstrued as limiting the scope thereof.

[0320] In the disclosure which follows, the following abbreviationsapply:° C. (degrees Centigrade); g (gravitational field); vol (volume);w/v (weight to volume); v/v (volume to volume); BSA (bovine serumalbumin); CTAB (cetyltrimethylammonium bromide); HPLC (high pressureliquid chromatography); DNA (deoxyribonucleic acid); p (plasmid); μl(microliters); ml (milliliters); μg (micrograms); pmoles (picomoles); mg(milligrams); M (molar); mM (milliMolar); μM (microMolar); nm(nanometers); kdal (kilodaltons); OD (optical density); EDTA (ethylenediamine tetra-acetic acid); FITC (fluorescein isothiocyanate); SDS(sodium dodecyl sulfate); NaPO₄ (sodium phosphate); Tris(tris(hydroxymethyl)-aminomethane); PMSF (phenylmethylsulfonylfluoride);TBE (Tris-Borate-EDTA, i.e., Tris buffer titrated with boric acid ratherthan HCl and containing EDTA); PBS (phosphate buffered saline); PPBS(phosphate buffered saline containing 1 mM PMSF); PAGE (polyacrylamidegel electrophoresis); Tween (polyoxyethylene-sorbitan); Dynal (DynalA.S., Oslo, Norway); Epicentre (Epicentre Technologies, Madison, Wis.);MJ Research (MJ Research, Watertown, Mass.); National Biosciences(Plymouth, Minn.); New England Biolabs (Beverly, Mass.); Novagen(Novagen, Inc., Madison, Wis.); Perkin Elmer (Norwalk, Conn.); PromegaCorp. (Madison, Wis.); Stratagene (Stratagene Cloning Systems, La Jolla,Calif.); USB (U.S. Biochemical, Cleveland, Ohio).

EXAMPLE 1 Characteristics of Native Thermostable DNA Polymerases

[0321] A. 5′ Nuclease Activity of DNAPTaq

[0322] During the polymerase chain reaction (PCR) [Saiki et al., Science239:487 (1988); Mullis and Faloona, Methods in Enzymology 155:335(1987)], DNAPTaq is able to amplify many, but not all, DNA sequences.One sequence that cannot be amplified using DNAPTaq is shown in FIG. 6(Hairpin structure is SEQ ID NO:15, PRIMERS are SEQ ID NOS:16-17.) ThisDNA sequence has the distinguishing characteristic of being able to foldon itself to form a hairpin with two single-stranded arms, whichcorrespond to the primers used in PCR.

[0323] To test whether this failure to amplify is due to the 5′ nucleaseactivity of the enzyme, we compared the abilities of DNAPTaq and DNAPStfto amplify this DNA sequence during 30 cycles of PCR. Syntheticoligonucleotides were obtained from The Biotechnology Center at theUniversity of Wisconsin-Madison. The DNAPTaq and DNAPStf were fromPerkin Elmer (i.e., Amplitaq™ DNA polymerase and the Stoffel fragment ofAmplitaq™ DNA polymerase). The substrate DNA comprised the hairpinstructure shown in FIG. 6 cloned in a double-stranded form into pUC19.The primers used in the amplification are listed as SEQ ID NOS:16-17.Primer SEQ ID NO:17 is shown annealed to the 3′ arm of the hairpinstructure in FIG. 6. Primer SEQ ID NO:16 is shown as the first 20nucleotides in bold on the 5′ arm of the hairpin in FIG. 6.

[0324] Polymerase chain reactions comprised 1 ng of supercoiled plasmidtarget DNA, 5 pmoles of each primer, 40 μM each dNTP, and 2.5 units ofDNAPTaq or DNAPStf, in a 50 μl solution of 10 mM Tris-Cl pH 8.3. TheDNAPTaq reactions included 50 μM KCl and 1.5 mM MgCl₂. The temperatureprofile was 95° C. for 30 sec., 55° C. for 11 min. and 72° C. for 1min., through 30 cycles. Ten percent of each reaction was analyzed bygel electrophoresis through 6% polyacrylamide (cross-linked 29:1) in abuffer of 45 mM Tris.Borate, pH 8.3, 1.4 mM EDTA.

[0325] The results are shown in FIG. 7. The expected product was made byDNAPStf (indicated simply as “S”) but not by DNAPTaq (indicated as “T”).We conclude that the 5′ nuclease activity of DNAPTaq is responsible forthe lack of amplification of this DNA sequence.

[0326] To test whether the 5′ unpaired nucleotides in the substrateregion of this structured DNA are removed by DNAPTaq, the fate of theend-labeled 5′ arm during four cycles of PCR was compared using the sametwo polymerases (FIG. 8). The hairpin templates, such as the onedescribed in FIG. 6, were made using DNAPStf and a ³²P-5′-end-labeledprimer. The 5′-end of the DNA was released as a few large fragments byDNAPTaq but not by DNAPStf. The sizes of these fragments (based on theirmobilities) show that they contain most or all of the unpaired 5′ arm ofthe DNA. Thus, cleavage occurs at or near the base of the bifurcatedduplex. These released fragments terminate with 3′ OH groups, asevidenced by direct sequence analysis, and the abilities of thefragments to be extended by terminal deoxynucleotidyl transferase.

[0327] FIGS. 9-11 show the results of experiments designed tocharacterize the cleavage reaction catalyzed by DNAPTaq. Unlessotherwise specified, the cleavage reactions comprised 0.01 pmoles ofheat-denatured, end-labeled hairpin DNA (with the unlabeledcomplementary strand also present), 1 pmole primer (complementary to the3′ arm) and 0.5 units of DNAPTaq (estimated to be 0.026 pmoles) in atotal volume of 10 μl of 10 mM Tris-Cl, ph 8.5, 50 mM KCl and 1.5 mMMgCl₂. As indicated, some reactions had different concentrations of KCl,and the precise times and temperatures used in each experiment areindicated in the individual figures. The reactions that included aprimer used the one shown in FIG. 6 (SEQ ID NO:17). In some instances,the primer was extended to the junction site by providing polymerase andselected nucleotides.

[0328] Reactions were initiated at the final reaction temperature by theaddition of either the MgCl₂ or enzyme. Reactions were stopped at theirincubation temperatures by the addition of 8 μl of 95% formamide with 20mM EDTA and 0.05% marker dyes. The Tm calculations listed were madeusing the Oligo™ primer analysis software from National Biosciences,Inc. These were determined using 0.25 μM as the DNA concentration, ateither 15 or 65 mM total salt (the 1.5 mM MgCl₂ in all reactions wasgiven the value of 15 mM salt for these calculations).

[0329]FIG. 9 is an autoradiogram containing the results of a set ofexperiments and conditions on the cleavage site. FIG. 9A is adetermination of reaction components that enable cleavage. Incubation of5′-end-labeled hairpin DNA was for 30 minutes at 55° C., with theindicated components. The products were resolved by denaturingpolyacrylamide gel electrophoresis and the lengths of the products, innucleotides, are indicated. FIG. 9B describes the effect of temperatureon the site of cleavage in the absence of added primer. Reactions wereincubated in the absence of KCl for 10 minutes at the indicatedtemperatures. The lengths of the products, in nucleotides, areindicated.

[0330] Surprisingly, cleavage by DNAPTaq requires neither a primer nordNTPs (see FIG. 9A). Thus, the 5′ nuclease activity can be uncoupledfrom polymerization. Nuclease activity requires magnesium ions, thoughmanganese ions can be substituted, albeit with potential changes inspecificity and activity. Neither zinc nor calcium ions support thecleavage reaction. The reaction occurs over a broad temperature range,from 25° C. to 85° C., with the rate of cleavage increasing at highertemperatures.

[0331] Still referring to FIG. 9, the primer is not elongated in theabsence of added dNTPs. However, the primer influences both the site andthe rate of cleavage of the hairpin. The change in the site of cleavage(FIG. 9A) apparently results from disruption of a short duplex formedbetween the arms of the DNA substrate. In the absence of primer, thesequences indicated by underlining in FIG. 6 could pair, forming anextended duplex. Cleavage at the end of the extended duplex wouldrelease the 11 nucleotide fragment seen on the FIG. 9A lanes with noadded primer. Addition of excess primer (FIG. 9A, lanes 3 and 4) orincubation at an elevated temperature (FIG. 9B) disrupts the shortextension of the duplex and results in a longer 5′ arm and, hence,longer cleavage products.

[0332] The location of the 3′ end of the primer can influence theprecise site of cleavage. Electrophoretic analysis revealed that in theabsence of primer (FIG. 9B), cleavage occurs at the end of the substrateduplex (either the extended or shortened form, depending on thetemperature) between the first and second base pairs. When the primerextends up to the base of the duplex, cleavage also occurs onenucleotide into the duplex. However, when a gap of four or sixnucleotides exists between the 3′ end of the primer and the substrateduplex, the cleavage site is shifted four to six nucleotides in the 5′direction.

[0333]FIG. 10 describes the kinetics of cleavage in the presence (FIG.10A) or absence (FIG. 10B) of a primer oligonucleotide. The reactionswere run at 55° C. with either 50 mM KCl (FIG. 10A) or 20 mM KCl (FIG.10B). The reaction products were resolved by denaturing polyacrylamidegel electrophoresis and the lengths of the products, in nucleotides, areindicated. “M”, indicating a marker, is a 5′ end-labeled 19-ntoligonucleotide. Under these salt conditions, FIGS. 10A and 10B indicatethat the reaction appears to be about twenty times faster in thepresence of primer than in the absence of primer. This effect on theefficiency may be attributable to proper alignment and stabilization ofthe enzyme on the substrate.

[0334] The relative influence of primer on cleavage rates becomes muchgreater when both reactions are run in 50 mM KCl. In the presence ofprimer, the rate of cleavage increases with KCl concentration, up toabout 50 mM. However, inhibition of this reaction in the presence ofprimer is apparent at 100 mM and is complete at 150 mM KCl. In contrast,in the absence of primer the rate is enhanced by concentration of KCl upto 20 mM, but it is reduced at concentrations above 30 mM. At 50 mM KCl,the reaction is almost completely inhibited. The inhibition of cleavageby KCl in the absence of primer is affected by temperature, being morepronounced at lower temperatures.

[0335] Recognition of the 5′ end of the arm to be cut appears to be animportant feature of substrate recognition. Substrates that lack a free5′ end, such as circular M13 DNA, cannot be cleaved under any conditionstested. Even with substrates having defined 5′ arms, the rate ofcleavage by DNAPTaq is influenced by the length of the arm. In thepresence of primer and 50 mM KCl, cleavage of a 5′ extension that is 27nucleotides long is essentially complete within 2 minutes at 55° C. Incontrast, cleavages of molecules with 5′ arms of 84 and 188 nucleotidesare only about 90% and 40% complete after 20 minutes. Incubation athigher temperatures reduces the inhibitory effects of long extensionsindicating that secondary structure in the 5′ arm or a heat-labilestructure in the enzyme may inhibit the reaction. A mixing experiment,run under conditions of substrate excess, shows that the molecules withlong arms do not preferentially tie up the available enzyme innon-productive complexes. These results may indicate that the 5′nuclease domain gains access to the cleavage site at the end of thebifurcated duplex by moving down the 5′ arm from one end to the other.Longer 5′ arms would be expected to have more adventitious secondarystructures (particularly when KCl concentrations are high), which wouldbe likely to impede this movement.

[0336] Cleavage does not appear to be inhibited by long 3′ arms ofeither the substrate strand target molecule or pilot nucleic acid, atleast up to 2 kilobases. At the other extreme, 3′ arms of the pilotnucleic acid as short as one nucleotide can support cleavage in aprimer-independent reaction, albeit inefficiently. Fully pairedoligonucleotides do not elicit cleavage of DNA templates during primerextension.

[0337] The ability of DNAPTaq to cleave molecules even when thecomplementary strand contains only one unpaired 3′ nucleotide may beuseful in optimizing allele-specific PCR. PCR primers that have unpaired3′ ends could act as pilot oligonucleotides to direct selective cleavageof unwanted templates during preincubation of potential template-primercomplexes with DNAPTaq in the absence of nucleoside triphosphates.

[0338] B. 5′ Nuclease Activities of Other DNAPs

[0339] To determine whether other 5′ nucleases in other DNAPs would besuitable for the present invention, an array of enzymes, several ofwhich were reported in the literature to be free of apparent 5′ nucleaseactivity, were examined. The ability of these other enzymes to cleavenucleic acids in a structure-specific manner was tested using thehairpin substrate shown in FIG. 6 under conditions reported to beoptimal for synthesis by each enzyme.

[0340] DNAPEc1 and DNAP Klenow were obtained from Promega Corporation;the DNAP of Pyrococcus furious [“Pfu”, Bargseid et al., Strategies 4:34(1991)] was from Strategene; the DNAP of Thermococcus litoralis [“Tli”,Vent™(exo−), Perler et al., Proc. Natl. Acad. Sci. USA 89:5577 (1992)]was from New England Biolabs; the DNAP of Thermus flavus [“Tfl”, Kaledinet al., Biokhimiya 46:1576 (1981)] was from Epicentre Technologies; andthe DNAP of Thermus thermophilus [“Tth”, Carballeira et al.,Biotechniques 9:276 (1990); Myers et al., Biochem. 30:7661 (1991)] wasfrom U.S. Biochemicals.

[0341] 0.5 units of each DNA polymerase was assayed in a 20 μl reaction,using either the buffers supplied by the manufacturers for theprimer-dependent reactions, or 10 mM Tris.Cl, pH 8.5, 1.5 mM MgCl₂, and20 mM KCl. Reaction mixtures were at held 72° C. before the addition ofenzyme.

[0342]FIG. 11 is an autoradiogram recording the results of these tests.FIG. 11A demonstrates reactions of endonucleases of DNAPs of severalthermophilic bacteria. The reactions were incubated at 55° C. for 10minutes in the presence of primer or at 72° C. for 30 minutes in theabsence of primer, and the products were resolved by denaturingpolyacrylamide gel electrophoresis. The lengths of the products, innucleotides, are indicated. FIG. 11B demonstrates endonucleolyticcleavage by the 5′ nuclease of DNAPEc1. The DNAPEc1 and DNAP Klenowreactions were incubated for 5 minutes at 37° C. Note the light band ofcleavage products of 25 and 11 nucleotides in the DNAPEc1 lanes (made inthe presence and absence of primer, respectively). FIG. 7B alsodemonstrates DNAPTaq reactions in the presence (+) or absence (−) ofprimer. These reactions were run in 50 mM and 20 mM KCl, respectively,and were incubated at 55° C. for 10 minutes.

[0343] Referring to FIG. 11A, DNAPs from the eubacteria Thermusthermophilus and Thermus flavus cleave the substrate at the same placeas DNAPTaq, both in the presence and absence of primer. In contrast,DNAPs from the archaebacteria Pyrococcus furiosus and Thermococcuslitoralis are unable to cleave the substrates endonucleolytically. TheDNAPs from Pyrococcus furious and Thermococcus litoralis share littlesequence homology with eubacterial enzymes (Ito et al., Nucl. Acids Res.19:4045 (1991); Mathur et al., Nucl. Acids. Res. 19:6952 (1991); seealso Perler et al.). Referring to FIG. 11B, DNAPEc1 also cleaves thesubstrate, but the resulting cleavage products are difficult to detectunless the 3′ exonuclease is inhibited. The amino acid sequences of the5′ nuclease domains of DNAPEc1 and DNAPTaq are about 38% homologous(Gelfand, supra).

[0344] The 5′ nuclease domain of DNAPTaq also shares about 19% homologywith the 5′ exonuclease encoded by gene 6 of bacteriophage T7 [Dunn etal., J. Mol. Biol. 166:477 (1983)]. This nuclease, which is notcovalently attached to a DNAP polymerization domain, is also able tocleave DNA endonucleolytically, at a site similar or identical to thesite that is cut by the 5′ nucleases described above, in the absence ofadded primers.

[0345] C. Transcleavage

[0346] The ability of a 5′ nuclease to be directed to cleave efficientlyat any specific sequence was demonstrated in the following experiment. Apartially complementary oligonucleotide termed a “pilot oligonucleotide”was hybridized to sequences at the desired point of cleavage. Thenon-complementary part of the pilot oligonucleotide provided a structureanalogous to the 3′ arm of the template (see FIG. 6), whereas the 5′region of the substrate strand became the 5′ arm. A primer was providedby designing the 3′ region of the pilot so that it would fold on itselfcreating a short hairpin with a stabilizing tetra-loop [Antao et al.,Nucl. Acids Res. 19:5901 (1991)]. Two pilot oligonucleotides are shownin FIG. 12A. Oligonucleotides 19-12 (SEQ ID NO:18), 30-12 (SEQ ID NO:19)and 30-0 (SEQ ID NO:20) are 31, 42 or 30 nucleotides long, respectively.However, oligonucleotides 19-12 (SEQ ID NO:18) and 34-19 (SEQ ID NO:19)have only 19 and 30 nucleotides, respectively, that are complementary todifferent sequences in the substrate strand. The pilot oligonucleotidesare calculated to melt off their complements at about 50° C. (19-12) andabout 75° C. (30-12). Both pilots have 12 nucleotides at their 3′ ends,which act as 3′ arms with base-paired primers attached.

[0347] To demonstrate that cleavage could be directed by a pilotoligonucleotide, we incubated a single-stranded target DNA with DNAPTaqin the presence of two potential pilot oligonucleotides. Thetranscleavage reactions, where the target and pilot nucleic acids arenot covalently linked, includes 0.01 pmoles of single end-labeledsubstrate DNA, I unit of DNAPTaq and 5 pmoles of pilot oligonucleotidein a volume of 20 μl of the same buffers. These components were combinedduring a one minute incubation at 95° C., to denature the PCR-generateddouble-stranded substrate DNA, and the temperatures of the reactionswere then reduced to their final incubation temperatures.Oligonucleotides 30-12 and 19-12 can hybridize to regions of thesubstrate DNAs that are 85 and 27 nucleotides from the 5′ end of thetargeted strand.

[0348]FIG. 21 shows the complete 206-mer sequence (SEQ ID NO:32). The206-mer was generated by PCR. The M13/pUC 24-mer reverse sequencing(−48) primer and the M13/pUC sequencing (−47) primer from New EnglandBiolabs (catalogue nos. 1233 and 1224 respectively) were used (50 pmoleseach) with the pGEM3z(f+) plasmid vector (Promega Corp.) as template (10ng) containing the target sequences. The conditions for PCR were asfollows: 50 μM of each dNTP and 2.5 units of Taq DNA polymerase in 100μl of 20 mM Tris-Cl, pH 8.3, 1.5 mM MgCl₂, 50 mM KCl with 0.05% Tween-20and 0.05% NP-40. Reactions were cycled 35 times through 95° C. for 45seconds, 63° C. for 45 seconds, then 72° C. for 75 seconds. Aftercycling, reactions were finished off with an incubation at 72° C. for 5minutes. The resulting fragment was purified by electrophoresis througha 6% polyacrylamide gel (29:1 cross link) in a buffer of 45 mMTris-Borate, pH 8.3, 1.4 mM EDTA, visualized by ethidium bromidestaining or autoradiography, excised from the gel, eluted by passivediffusion, and concentrated by ethanol precipitation.

[0349] Cleavage of the substrate DNA occurred in the presence of thepilot oligonucleotide 19-12 at 50° C. (FIG. 12B, lanes 1 and 7) but notat 75° C. (lanes 4 and 10). In the presence of oligonucleotide 30-12cleavage was observed at both temperatures. Cleavage did not occur inthe absence of added oligonucleotides (lanes 3, 6 and 12) or at about80° C. even though at 50° C. adventitious structures in the substrateallowed primer-independent cleavage in the absence of KCl (FIG. 12B,lane 9). A non-specific oligonucleotide with no complementarity to thesubstrate DNA did not direct cleavage at 50° C., either in the absenceor presence of 50 mM KCl (lanes 13 and 14). Thus, the specificity of thecleavage reactions can be controlled by the extent of complementarity tothe substrate and by the conditions of incubation.

[0350] D. Cleavage of RNA

[0351] An shortened RNA version of the sequence used in thetranscleavage experiments discussed above was tested for its ability toserve as a substrate in the reaction. The RNA is cleaved at the expectedplace, in a reaction that is dependent upon the presence of the pilotoligonucleotide. The RNA substrate, made by T7 RNA polymerase in thepresence of [α-³²P]UTP, corresponds to a truncated version of the DNAsubstrate used in FIG. 12B. Reaction conditions were similar to those inused for the DNA substrates described above, with 50 mM KCl; incubationwas for 40 minutes at 55° C. The pilot oligonucleotide used is termed30-0 (SEQ ID NO:20) and is shown in FIG. 13A.

[0352] The results of the cleavage reaction is shown in FIG. 13B. Thereaction was run either in the presence or absence of DNAPTaq or pilotoligonucleotide as indicated in FIG. 13B.

[0353] Strikingly, in the case of RNA cleavage, a 3′ arm is not requiredfor the pilot oligonucleotide. It is very unlikely that this cleavage isdue to previously described RNaseH, which would be expected to cut theRNA in several places along the 30 base-pair long RNA-DNA duplex. The 5′nuclease of DNAPTaq is a structure-specific RNaseH that cleaves the RNAat a single site near the 5′ end of the heteroduplexed region.

[0354] It is surprising that an oligonucleotide lacking a 3′ arm is ableto act as a pilot in directing efficient cleavage of an RNA targetbecause such oligonucleotides are unable to direct efficient cleavage ofDNA targets using native DNAPs. However, some 5′ nucleases of thepresent invention (for example, clones E, F and G of FIG. 4) can cleaveDNA in the absence of a 3′ arm. In other words, a non-extendablecleavage structure is not required for specific cleavage with some 5′nucleases of the present invention derived from thermostable DNApolymerases.

[0355] We tested whether cleavage of an RNA template by DNAPTaq in thepresence of a fully complementary primer could help explain why DNAPTaqis unable to extend a DNA oligonucleotide on an RNA template, in areaction resembling that of reverse transcriptase. Another thermophilicDNAP, DNAPTth, is able to use RNA as a template, but only in thepresence of Mn++, so we predicted that this enzyme would not cleave RNAin the presence of this cation. Accordingly, we incubated an RNAmolecule with an appropriate pilot oligonucleotide in the presence ofDNAPTaq or DNAPTth, in buffer containing either Mg++ or Mn++. Asexpected, both enzymes cleaved the RNA in the presence of Mg++. However,DNAPTaq, but not DNAPTth, degraded the RNA in the presence of Mn++. Weconclude that the 5′ nuclease activities of many DNAPs may contribute totheir inability to use RNA as templates.

EXAMPLE 2 Generation of 5′ Nucleases from Thermostable DNA Polymerases

[0356] Thermostable DNA polymerases were generated which have reducedsynthetic activity, an activity that is an undesirable side-reactionduring DNA cleavage in the detection assay of the invention, yet havemaintained thermostable nuclease activity. The result is a thermostablepolymerase which cleaves nucleic acids DNA with extreme specificity.

[0357] Type A DNA polymerases from eubacteria of the genus Thermus shareextensive protein sequence identity (90% in the polymerization domain,using the Lipman-Pearson method in the DNA analysis software fromDNAStar, Wis.) and behave similarly in both polymerization and nucleaseassays. Therefore, we have used the genes for the DNA polymerase ofThermus aquaticus (DNAPTaq) and Thermus flavus (DNAPTfl) asrepresentatives of this class. Polymerase genes from other eubacterialorganisms, such as Thermus thermophilus, Thermus sp., Thermotogamaritima, Thermosipho africanus and Bacillus stearothermophilus areequally suitable. The DNA polymerases from these thermophilic organismsare capable of surviving and performing at elevated temperatures, andcan thus be used in reactions in which temperature is used as aselection against non-specific hybridization of nucleic acid strands.

[0358] The restriction sites used for deletion mutagenesis, describedbelow, were chosen for convenience. Different sites situated withsimilar convenience are available in the Thermus thermophilus gene andcan be used to make similar constructs with other Type A polymerasegenes from related organisms.

[0359] A. Creation of 5′ Nuclease Constructs

[0360] 1. Modified DNAPTaq Genes

[0361] The first step was to place a modified gene for the Taq DNApolymerase on a plasmid under control of an inducible promoter. Themodified Taq polymerase gene was isolated as follows: The Taq DNApolymerase gene was amplified by polymerase chain reaction from genomicDNA from Thermus aquaticus, strain YT-1 (Lawyer et al., supra), using asprimers the oligonucleotides described in SEQ ID NOS:13-14. Theresulting fragment of DNA has a recognition sequence for the restrictionendonuclease EcoRI at the 5′ end of the coding sequence and a BglIIsequence at the 3′ end. Cleavage with BglII leaves a 5′ overhang or“sticky end” that is compatible with the end generated by BamHI. ThePCR-amplified DNA was digested with EcoRI and BamHI. The 2512 bpfragment containing the coding region for the polymerase gene was gelpurified and then ligated into a plasmid which contains an induciblepromoter.

[0362] In one embodiment of the invention, the pTTQ18 vector, whichcontains the hybrid trp-lac (tac) promoter, was used [M.J.R. Stark, Gene5:255 (1987)] and shown in FIG. 14. The tac promoter is under thecontrol of the E. coli lac repressor. Repression allows the synthesis ofthe gene product to be suppressed until the desired level of bacterialgrowth has been achieved, at which point repression is removed byaddition of a specific inducer, isopropyl-β-D-thiogalactopyranoside(IPTG). Such a system allows the expression of foreign proteins that mayslow or prevent growth of transformants.

[0363] Bacterial promoters, such as tac, may not be adequatelysuppressed when they are present on a multiple copy plasmid. If a highlytoxic protein is placed under control of such a promoter, the smallamount of expression leaking through can be harmful to the bacteria. Inanother embodiment of the invention, another option for repressingsynthesis of a cloned gene product was used. The non-bacterial promoter,from bacteriophage T7, found in the plasmid vector series pET-3 was usedto express the cloned mutant Taq polymerase genes [FIG. 15; Studier andMoffatt, J. Mol. Biol. 189:113 (1986)]. This promoter initiatestranscription only by T7 RNA polymerase. In a suitable strain, such asBL21(DE3)pLYS, the gene for this RNA polymerase is carried on thebacterial genome under control of the lac operator. This arrangement hasthe advantage that expression of the multiple copy gene (on the plasmid)is completely dependent on the expression of T7 RNA polymerase, which iseasily suppressed because it is present in a single copy.

[0364] For ligation into the pTTQ18 vector (FIG. 14), the PCR productDNA containing the Taq polymerase coding region (mutTaq, clone 4B, SEQID NO:21) was digested with EcoRI and BglII and this fragment wasligated under standard “sticky end” conditions [Sambrook et al.Molecular Cloning, Cold Spring Harbor Laboratory Press, Cold SpringHarbor, pp. 1.63-1.69 (1989)] into the EcoRI and BamHI sites of theplasmid vector pTTQ18. Expression of this construct yields atranslational fusion product in which the first two residues of thenative protein (Met-Arg) are replaced by three from the vector(Met-Asn-Ser), but the remainder of the natural protein would notchange. The construct was transformed into the JM109 strain of E. coliand the transformants were plated under incompletely repressingconditions that do not permit growth of bacteria expressing the nativeprotein. These plating conditions allow the isolation of genescontaining pre-existing mutations, such as those that result from theinfidelity of Taq polymerase during the amplification process.

[0365] Using this amplification/selection protocol, we isolated a clone(depicted in FIG. 4B) containing a mutated Taq polymerase gene (mutTaq,clone 4B). The mutant was first detected by its phenotype, in whichtemperature-stable 5′ nuclease activity in a crude cell extract wasnormal, but polymerization activity was almost absent (approximatelyless than 1% of wild type Taq polymerase activity).

[0366] DNA sequence analysis of the recombinant gene showed that it hadchanges in the polymerase domain resulting in two amino acidsubstitutions: an A to G change at nucleotide position 1394 causes a Gluto Gly change at amino acid position 465 (numbered according to thenatural nucleic and amino acid sequences, SEQ ID NOS:1 and 4) andanother A to G change at nucleotide position 2260 causes a Gln to Argchange at amino acid position 754. Because the Gin to Gly mutation is ata nonconserved position and because the Glu to Arg mutation alters anamino acid that is conserved in virtually all of the known Type Apolymerases, this latter mutation is most likely the one responsible forcurtailing the synthesis activity of this protein. The nucleotidesequence for the FIG. 4B construct is given in SEQ ID NO:21. The enzymeencoded by this sequence is referred to as Cleavase® A/G.

[0367] Subsequent derivatives of DNAPTaq constructs were made from themutTaq gene, thus, they all bear these amino acid substitutions inaddition to their other alterations, unless these particular regionswere deleted. These mutated sites are indicated by black boxes at theselocations in the diagrams in FIG. 4. In FIG. 4, the designation “3′ Exo”is used to indicate the location of the 3′ exonuclease activityassociated with Type A polymerases which is not present in DNAPTaq. Allconstructs except the genes shown in FIGS. 4E, F and G were made in thepTTQ18 vector.

[0368] The cloning vector used for the genes in FIGS. 4E and F was fromthe commercially available pET-3 series, described above. Though thisvector series has only a BamHI site for cloning downstream of the T7promoter, the series contains variants that allow cloning into any ofthe three reading frames. For cloning of the PCR product describedabove, the variant called pET-3c was used (FIG. 15). The vector wasdigested with BamHI, dephosphorylated with calf intestinal phosphatase,and the sticky ends were filled in using the Klenow fragment of DNAPEc1and dNTPs. The gene for the mutant Taq DNAP shown in FIG. 4B (mutTaq,clone 4B) was released from pTTQ18 by digestion with EcoRI and SalI, andthe “sticky ends” were filled in as was done with the vector. Thefragment was ligated to the vector under standard blunt-end conditions(Sambrook et al., Molecular Cloning, supra), the construct wastransformed into the BL21(DE3)pLYS strain of E. coli, and isolates werescreened to identify those that were ligated with the gene in the properorientation relative to the promoter. This construction yields anothertranslational fusion product, in which the first two amino acids ofDNAPTaq (Met-Arg) are replaced by 13 from the vector plus two from thePCR primer (Met-Ala-Ser-Met-Thr-Gly-Gly-Gln-Gln-Met-Gly-Arg-Ile-Asn-Ser)(SEQ ID NO:29).

[0369] Our goal was to generate enzymes that lacked the ability tosynthesize DNA, but retained the ability to cleave nucleic acids with a5′ nuclease activity. The act of primed, templated synthesis of DNA isactually a coordinated series of events, so it is possible to disableDNA synthesis by disrupting one event while not affecting the others.These steps include, but are not limited to, primer recognition andbinding, dNTP binding and catalysis of the inter-nucleotidephosphodiester bond. Some of the amino acids in the polymerizationdomain of DNAPEcI have been linked to these functions, but the precisemechanisms are as yet poorly defined.

[0370] One way of destroying the polymerizing ability of a DNApolymerase is to delete all or part of the gene segment that encodesthat domain for the protein, or to otherwise render the gene incapableof making a complete polymerization domain. Individual mutant enzymesmay differ from each other in stability and solubility both inside andoutside cells. For instance, in contrast to the 5′ nuclease domain ofDNAPEcI, which can be released in an active form from the polymerizationdomain by gentle proteolysis [Setlow and Kornberg, J. Biol. Chem.247:232 (1972)], the Thermus nuclease domain, when treated similarly,becomes less soluble and the cleavage activity is often lost.

[0371] Using the mutant gene shown in FIG. 4B as starting material,several deletion constructs were created. All cloning technologies werestandard (Sambrook et al., supra) and are summarized briefly, asfollows:

[0372]FIG. 4C: The mutTaq construct was digested with PstI, which cutsonce within the polymerase coding region, as indicated, and cutsimmediately downstream of the gene in the multiple cloning site of thevector. After release of the fragment between these two sites, thevector was re-ligated, creating an 894-nucleotide deletion, and bringinginto frame a stop codon 40 nucleotides downstream of the junction. Thenucleotide sequence of this 5′ nuclease (clone 4C) is given in SEQ IDNO:9.

[0373]FIG. 4D: The mutTaq construct was digested with NheI, which cutsonce in the gene at position 2047. The resulting four-nucleotide 5′overhanging ends were filled in, as described above, and the blunt endswere re-ligated. The resulting four-nucleotide insertion changes thereading frame and causes termination of translation ten amino acidsdownstream of the mutation. The nucleotide sequence of this 5′ nuclease(clone 4D) is given in SEQ ID NO:10.

[0374]FIG. 4E: The entire mutTaq gene was cut from pTTQ18 using EcoRIand SalI and cloned into pET-3c, as described above. This clone wasdigested with BstXI and XcmI, at unique sites that are situated as shownin FIG. 4E. The DNA was treated with the Klenow fragment of DNAPEc1 anddNTPs, which resulted in the 3′ overhangs of both sites being trimmed toblunt ends. These blunt ends were ligated together, resulting in anout-of-frame deletion of 1540 nucleotides. An in-frame termination codonoccurs 18 triplets past the junction site. The nucleotide sequence ofthis 5′ nuclease (clone 4E) is given in SEQ ID NO:11, with theappropriate leader sequence given in SEQ ID NO:30. It is also referredto as Cleavase® BX.

[0375]FIG. 4F: The entire mutTaq gene was cut from pTTQ18 using EcoRIand SalI and cloned into pET-3c, as described above. This clone wasdigested with BstXI and BamHI, at unique sites that are situated asshown in the diagram. The DNA was treated with the Klenow fragment ofDNAPEc1 and dNTPs, which resulted in the 3′ overhang of the BstXI sitebeing trimmed to a blunt end, while the 5′ overhang of the BamHI sitewas filled in to make a blunt end. These ends were ligated together,resulting in an in-frame deletion of 903 nucleotides. The nucleotidesequence of the 5′ nuclease (clone 4F) is given in SEQ ID NO:12. It isalso referred to as Cleavase® BB.

[0376]FIG. 4G: This polymerase is a variant of that shown in FIG. 4E. Itwas cloned in the plasmid vector pET-21 (Novagen). The non-bacterialpromoter from bacteriophage T7, found in this vector, initiatestranscription only by T7 RNA polymerase. See Studier and Moffatt, supra.In a suitable strain, such as (DES)pLYS, the gene for this RNApolymerase is carried on the bacterial genome under control of the lacoperator. This arrangement has the advantage that expression of themultiple copy gene (on the plasmid) is completely dependent on theexpression of T7 RNA polymerase, which is easily suppressed because itis present in a single copy. Because the expression of these mutantgenes is under this tightly controlled promoter, potential problems oftoxicity of the expressed proteins to the host cells are less of aconcern.

[0377] The pET-21 vector also features a “His*Tag”, a stretch of sixconsecutive histidine residues that are added on the carboxy terminus ofthe expressed proteins. The resulting proteins can then be purified in asingle step by metal chelation chromatography, using a commericallyavailable (Novagen) column resin with immobilized Ni⁺⁺ ions. The 2.5 mlcolumns are reusable, and can bind up to 20 mg of the target proteinunder native or denaturing (guanidine*HCl or urea) conditions.

[0378]E. coli (DES)pLYS cells are transformed with the constructsdescribed above using standard transformation techniques, and used toinoculate a standard growth medium (e.g., Luria-Bertani broth).Production of T7 RNA polymerase is induced during log phase growth byaddition of IPTG and incubated for a further 12 to 17 hours. Aliquots ofculture are removed both before and after induction and the proteins areexamined by SDS-PAGE. Staining with Coomassie Blue allows visualizationof the foreign proteins if they account for about 3-5% of the cellularprotein and do not co-migrate with any of the major protein bands.Proteins that co-migrate with major host protein must be expressed asmore than 10% of the total protein to be seen at this tage of analysis.

[0379] Some mutant proteins are sequestered by the cells into inclusionbodies. These are granules that form in the cytoplasm when bacteria aremade to express high levels of a foreign protein, and they can bepurified from a crude lysate, and analyzed by SDS-PAGE to determinetheir protein content. If the cloned protein is found in the inclusionbodies, it must be released to assay the cleavage and polymeraseactivities. Different methods of solubilization may be appropriate fordifferent proteins, and a variety of methods are known. See e.g.,Builder & Ogez, U.S. Pat. No. 4,511,502 (1985); Olson, U.S. Pat. No.4,518,526 (1985); Olson & Pai, U.S. Pat. No. 4,511,503 (1985); Jones etal., U.S. Pat. No. 4,512,922 (1985), all of which are herebyincorporated by reference.

[0380] The solubilized protein is then purified on the Ni⁺⁺ column asdescribed above, following the manufacturers instructions (Novagen). Thewashed proteins are eluted from the column by a combination of imidazolecompetitor (1 M) and high salt (0.5 M NaCl), and dialyzed to exchangethe buffer and to allow denature proteins to refold. Typical recoveriesresult in approximately 20 μg of specific protein per ml of startingculture. The DNAP mutant is referred to as Cleavase® BN and the sequenceis given in SEQ ID NO:31.

[0381] 2. Modified DNAPTfl Gene

[0382] The DNA polymerase gene of Thermus flavus was isolated from the“T. flavus” AT-62 strain obtained from the American Type TissueCollection (ATCC 33923). This strain has a different restriction mapthen does the T. flavus strain used to generate the sequence publishedby Akhmetzjanov and Vakhitov, supra. The published sequence is listed asSEQ ID NO:2. No sequence data has been published for the DNA polymerasegene from the AT-62 strain of T. flavus.

[0383] Genomic DNA from T. flavus was amplified using the same primersused to amplify the T. aquaticus DNA polymerase gene (SEQ ID NOS:13-14).The approximately 2500 base pair PCR fragment was digested with EcoRIand BamHI. The over-hanging ends were made blunt with the Klenowfragment of DNAPEc1 and dNTPs. The resulting approximately 1800 basepair fragment containing the coding region for the N-terminus wasligated into pET-3c, as described above. This construct, clone 5B, isdepicted in FIG. 5B. The wild type T. flavus DNA polymerase gene isdepicted in FIG. 5A. The SB clone has the same leader amino acids as dothe DNAPTaq clones 4E and F which were cloned into pET-3c; it is notknown precisely where translation termination occurs, but the vector hasa strong transcription termination signal immediately downstream of thecloning site.

[0384] B. Growth and Induction of Transformed Cells

[0385] Bacterial cells were transformed with the constructs describedabove using standard transformation techniques and used to inoculate 2mls of a standard growth medium (e.g., Luria-Bertani broth). Theresulting cultures were incubated as appropriate for the particularstrain used, and induced if required for a particular expression system.For all of the constructs depicted in FIGS. 4 and 5, the cultures weregrown to an optical density (at 600 nm wavelength) of 0.5 OD.

[0386] To induce expression of the cloned genes, the cultures werebrought to a final concentration of 0.4 mM IPTG and the incubations werecontinued for 12 to 17 hours. 50 μl aliquots of each culture wereremoved both before and after induction and were combined with 20 μl ofa standard gel loading buffer for sodium dodecyl sulfate-polyacrylamidegel electrophoresis (SDS-PAGE). Subsequent staining with Coomassie Blue(Sambrook et al., supra) allows visualization of the foreign proteins ifthey account for about 3-5% of the cellular protein and do notco-migrate with any of the major E. coli protein bands. Proteins that doco-migrate with a major host protein must be expressed as more than 10%of the total protein to be seen at this stage of analysis.

[0387] C. Heat Lysis and Fractionation

[0388] Expressed thermostable proteins, i.e., the 5′ nucleases, wereisolated by heating crude bacterial cell extracts to cause denaturationand precipitation of the less stable E. coli proteins. The precipitatedE. coli proteins were then, along with other cell debris, removed bycentrifugation. 1.7 mls of the culture were pelleted bymicrocentrifugation at 12,000 to 14,000 rpm for 30 to 60 seconds. Afterremoval of the supernatant, the cells were resuspended in 400 μl ofbuffer A (50 mM Tris-HCl, pH 7.9, 50 mM dextrose, 1 mM EDTA),re-centrifuged, then resuspended in 80 μl of buffer A with 4 mg/mllysozyme. The cells were incubated at room temperature for 15 minutes,then combined with 80 μl of buffer B (10 mM Tris-HCl, pH 7.9, 50 mM KCl,1 mM EDTA, 1 mM PMSF, 0.5% Tween-20, 0.5% Nonidet-P40).

[0389] This mixture was incubated at 75° C. for 1 hour to denature andprecipitate the host proteins. This cell extract was centrifuged at14,000 rpm for 15 minutes at 4° C., and the supernatant was transferredto a fresh tube. An aliquot of 0.5 to 1 μl of this supernatant was useddirectly in each test reaction, and the protein content of the extractwas determined by subjecting 7 μl to electrophoretic analysis, as above.The native recombinant Taq DNA polymerase [Englke, Anal. Biochem 191:396(1990)], and the double point mutation protein shown in FIG. 4B are bothsoluble and active at this point.

[0390] The foreign protein may not be detected after the heat treatmentsdue to sequestration of the foreign protein by the cells into inclusionbodies. These are granules that form in the cytoplasm when bacteria aremade to express high levels of a foreign protein, and they can bepurified from a crude lysate, and analyzed SDS PAGE to determine theirprotein content. Many methods have been described in the literature, andone approach is described below.

[0391] D. Isolation and Solubilization of Inclusion Bodies

[0392] A small culture was grown and induced as described above. A 1.7ml aliquot was pelleted by brief centrifugation, and the bacterial cellswere resuspended in 100 μl of Lysis buffer (50 mM Tris-HCl, pH 8.0, 1 mMEDTA, 100 mM NaCl). 2.5 μl of 20 mM PMSF were added for a finalconcentration of 0.5 mM, and lysozyme was added to a concentration of1.0 mg/ml. The cells were incubated at room temperature for 20 minutes,deoxycholic acid was added to 1 mg/ml (1 μl of 100 mg/ml solution), andthe mixture was further incubated at 37° C. for about 15 minutes oruntil viscous. DNAse I was added to 10 μg/ml and the mixture wasincubated at room temperature for about 30 minutes or until it was nolonger viscous.

[0393] From this mixture the inclusion bodies were collected bycentrifugation at 14,000 rpm for 15 minutes at 4° C., and thesupernatant was discarded. The pellet was resuspended in 100 μl of lysisbuffer with 10 mM EDTA (pH 8.0) and 0.5% Triton X-100. After 5 minutesat room temperature, the inclusion bodies were pelleted as before, andthe supernatant was saved for later analysis. The inclusion bodies wereresuspended in 50 μl of distilled water, and 5 μl was combined with SDSgel loading buffer (which dissolves the inclusion bodies) and analyzedelectrophoretically, along with an aliquot of the supernatant.

[0394] If the cloned protein is found in the inclusion bodies, it may bereleased to assay the cleavage and polymerase activities and the methodof solubilization must be compatible with the particular activity.Different methods of solubilization may be appropriate for differentproteins, and a variety of methods are discussed in Molecular Cloning(Sambrook et al., supra). The following is an adaptation we have usedfor several of our isolates.

[0395] 20 μl of the inclusion body-water suspension were pelleted bycentrifugation at 14,000 rpm for 4 minutes at room temperature, and thesupernatant was discarded. To further wash the inclusion bodies, thepellet was resuspended in 20 μl of lysis buffer with 2M urea, andincubated at room temperature for one hour. The washed inclusion bodieswere then resuspended in 2 μl of lysis buffer with 8M urea; the solutionclarified visibly as the inclusion bodies dissolved. Undissolved debriswas removed by centrifugation at 14,000 rpm for 4 minutes at roomtemperature, and the extract supernatant was transferred to a freshtube.

[0396] To reduce the urea concentration, the extract was diluted intoKH₂PO₄. A fresh tube was prepared containing 180 μl of 50 mM KH₂PO₄, pH9.5, 1 mM EDTA and 50 mM NaCl. A 2 μl aliquot of the extract was addedand vortexed briefly to mix. This step was repeated until all of theextract had been added for a total of 10 additions. The mixture wasallowed to sit at room temperature for 15 minutes, during which timesome precipitate often forms. Precipitates were removed bycentrifugation at 14,000 rpm, for 15 minutes at room temperature, andthe supernatant was transferred to a fresh tube. To the 200 μl ofprotein in the KH₂PO₄ solution, 140-200 μl of saturated (NH₄)₂SO₄ wereadded, so that the resulting mixture was about 41% to 50% saturated(NH₄)₂SO₄. The mixture was chilled on ice for 30 minutes to allow theprotein to precipitate, and the protein was then collected bycentrifugation at 14,000 rpm, for 4 minutes at room temperature. Thesupernatant was discarded, and the pellet was dissolved in 20 μl BufferC (20 mM HEPES, pH 7.9, 1 mM EDTA, 0.5% PMSF, 25 mM KCl and 0.5% each ofTween-20 and Nonidet P 40). The protein solution was centrifuged againfor 4 minutes to pellet insoluble materials, and the supernatant wasremoved to a fresh tube. The protein contents of extracts prepared inthis manner were visualized by resolving 1-4 μl by SDS-PAGE; 0.5 to 1 μlof extract was tested in the cleavage and polymerization assays asdescribed.

[0397] E. Protein Analysis for Presence of Nuclease and SyntheticActivity

[0398] The 5′ nucleases described above and shown in FIGS. 4 and 5 wereanalyzed by the following methods.

[0399] 1. Structure Specific Nuclease Assay

[0400] A candidate modified polymerase is tested for 5′ nucleaseactivity by examining its ability to catalyze structure-specificcleavages. By the term “cleavage structure” as used herein, is meant anucleic acid structure which is a substrate for cleavage by the 5′nuclease activity of a DNAP.

[0401] The polymerase is exposed to test complexes that have thestructures shown in FIG. 16. Testing for 5′ nuclease activity involvesthree reactions: 1) a primer-directed cleavage (FIG. 16B) is performedbecause it is relatively insensitive to variations in the saltconcentration of the reaction and can, therefore, be performed inwhatever solute conditions the modified enzyme requires for activity;this is generally the same conditions preferred by unmodifiedpolymerases; 2) a similar primer-directed cleavage is performed in abuffer which permits primer-independent cleavage, i.e., a low saltbuffer, to demonstrate that the enzyme is viable under these conditions;and 3) a primer-independent cleavage (FIG. 16A) is performed in the samelow salt buffer.

[0402] The bifurcated duplex is formed between a substrate strand and atemplate strand as shown in FIG. 16. By the term “substrate strand” asused herein, is meant that strand of nucleic acid in which the cleavagemediated by the 5′ nuclease activity occurs. The substrate strand isalways depicted as the top strand in the bifurcated complex which servesas a substrate for 5′ nuclease cleavage (FIG. 16). By the term “templatestrand” as used herein, is meant the strand of nucleic acid which is atleast partially complementary to the substrate strand and which annealsto the substrate strand to form the cleavage structure. The templatestrand is always depicted as the bottom strand of the bifurcatedcleavage structure (FIG. 16). If a primer (a short oligonucleotide of 19to 30 nucleotides in length) is added to the complex, as whenprimer-dependent cleavage is to be tested, it is designed to anneal tothe 3′ arm of the template strand (FIG. 16B). Such a primer would beextended along the template strand if the polymerase used in thereaction has synthetic activity.

[0403] The cleavage structure may be made as a single hairpin molecule,with the 3′ end of the target and the 5′ end of the pilot joined as aloop as shown in FIG. 16E. A primer oligonucleotide complementary to the3′ arm is also required for these tests so that the enzyme's sensitivityto the presence of a primer may be tested.

[0404] Nucleic acids to be used to form test cleavage structures can bechemically synthesized, or can be generated by standard recombinant DNAtechniques. By the latter method, the hairpin portion of the moleculecan be created by inserting into a cloning vector duplicate copies of ashort DNA segment, adjacent to each other but in opposing orientation.The double-stranded fragment encompassing this inverted repeat, andincluding enough flanking sequence to give short (about 20 nucleotides)unpaired 5′ and 3′ arms, can then be released from the vector byrestriction enzyme digestion, or by PCR performed with an enzyme lackinga 5′ exonuclease (e.g., the Stoffel fragment of Amplitaq™ DNApolymerase, Vent™ DNA polymerase).

[0405] The test DNA can be labeled on either end, or internally, witheither a radioisotope, or with a non-isotopic tag. Whether the hairpinDNA is a synthetic single strand or a cloned double strand, the DNA isheated prior to use to melt all duplexes. When cooled on ice, thestructure depicted in FIG. 16E is formed, and is stable for sufficienttime to perform these assays.

[0406] To test for primer-directed cleavage (Reaction 1), a detectablequantity of the test molecule (typically 1-100 fmol of ³²P-labeledhairpin molecule) and a 10 to 100-fold molar excess of primer are placedin a buffer known to be compatible with the test enzyme. For Reaction 2,where primer-directed cleavage is performed under condition which allowprimer-independent cleavage, the same quantities of molecules are placedin a solution that is the same as the buffer used in Reaction 1regarding pH, enzyme stabilizers (e.g., bovine serum albumin, nonionicdetergents, gelatin) and reducing agents (e.g., dithiothreitol,2-mercaptoethanol) but that replaces any monovalent cation salt with 20mM KCl; 20 mM KCl is the demonstrated optimum for primer-independentcleavage. Buffers for enzymes, such as DNAPEc1, that usually operate inthe absence of salt are not supplemented to achieve this concentration.To test for primer-independent cleavage (Reaction 3) the same quantityof the test molecule, but no primer, are combined under the same bufferconditions used for Reaction 2.

[0407] All three test reactions are then exposed to enough of the enzymethat the molar ratio of enzyme to test complex is approximately 1:1. Thereactions are incubated at a range of temperatures up to, but notexceeding, the temperature allowed by either the enzyme stability or thecomplex stability, whichever is lower, up to 80° C. for enzymes fromthermophiles, for a time sufficient to allow cleavage (10 to 60minutes). The products of Reactions 1, 2 and 3 are resolved bydenaturing polyacrylamide gel electrophoresis, and visualized byautoradiography or by a comparable method appropriate to the labelingsystem used. Additional labeling systems include chemiluminescencedetection, silver or other stains, blotting and probing and the like.The presence of cleavage products is indicated by the presence ofmolecules which migrate at a lower molecular weight than does theuncleaved test structure. These cleavage products indicate that thecandidate polymerase has structure-specific 5′ nuclease activity.

[0408] To determine whether a modified DNA polymerase has substantiallythe same 5′ nuclease activity as that of the native DNA polymerase, theresults of the above-described tests are compared with the resultsobtained from these tests performed with the native DNA polymerase. By“substantially the same 5′ nuclease activity” we mean that the modifiedpolymerase and the native polymerase will both cleave test molecules inthe same manner. It is not necessary that the modified polymerase cleaveat the same rate as the native DNA polymerase.

[0409] Some enzymes or enzyme preparations may have other associated orcontaminating activities that may be functional under the cleavageconditions described above and that may interfere with 5′ nucleasedetection. Reaction conditions can be modified in consideration of theseother activities, to avoid destruction of the substrate, or othermasking of the 5′ nuclease cleavage and its products. For example, theDNA polymerase I of E. Coli (Pol I), in addition to its polymerase and5′ nuclease activities, has a 3′ exonuclease that can degrade DNA in a3′ to 5′ direction. Consequently, when the molecule in FIG. 16E isexposed to this polymerase under the conditions described above, the 3′exonuclease quickly removes the unpaired 3′ arm, destroying thebifurcated structure required of a substrate for the 5′ exonucleasecleavage and no cleavage is detected. The true ability of Pol I tocleave the structure can be revealed if the 3′ exonuclease is inhibitedby a change of conditions (e.g., pH), mutation, or by addition of acompetitor for the activity. Addition of 500 pmoles of a single-strandedcompetitor oligonucleotide, unrelated to the FIG. 16E structure, to thecleavage reaction with Pol I effectively inhibits the digestion of the3′ arm of the FIG. 16E structure without interfering with the 5′exonuclease release of the 5′ arm. The concentration of the competitoris not critical, but should be high enough to occupy the 3′ exonucleasefor the duration of the reaction.

[0410] Similar destruction of the test molecule may be caused bycontaminants in the candidate polymerase preparation. Several sets ofthe structure specific nuclease reactions may be performed to determinethe purity of the candidate nuclease and to find the window betweenunder and over exposure of the test molecule to the polymerasepreparation being investigated.

[0411] The above described modified polymerases were tested for 5′nuclease activity as follows: Reaction I was performed in a buffer of 10mM Tris-Cl, pH 8.5 at 20° C., 1.5 mM MgCl₂ and 50 mM KCl and in Reaction2 the KCl concentration was reduced to 20 mM. In Reactions 1 and 2, 10fmoles of the test substrate molecule shown in FIG. 16E were combinedwith 1 pmole of the indicated primer and 0.5 to 1.0 μl of extractcontaining the modified polymerase (prepared as described above). Thismixture was then incubated for 10 minutes at 55° C. For all of themutant polymerases tested these conditions were sufficient to givecomplete cleavage. When the molecule shown in FIG. 16E was labeled atthe 5′ end, the released 5′ fragment, 25 nucleotides long, wasconveniently resolved on a 20% polyacrylamide gel (19:1 cross-linked)with 7 M urea in a buffer containing 45 mM Tris-borate pH 8.3, 1.4 mMEDTA. Clones 4C-F and 5B exhibited structure-specific cleavagecomparable to that of the unmodified DNA polymerase. Additionally,clones 4E, 4F and 4G have the added ability to cleave DNA in the absenceof a 3′ arm as discussed above. Representative cleavage reactions areshown in FIG. 17.

[0412] For the reactions shown in FIG. 17, the mutant polymerase clones4E (Taq mutant) and SB (Tfl mutant) were examined for their ability tocleave the hairpin substrate molecule shown in FIG. 16E. The substratemolecule was labeled at the 5′ terminus with ³²P, 10 fmoles ofheat-denatured, end-labeled substrate DNA and 0.5 units of DNAPTaq(lane 1) or 0.5 μl of 4e or 5b extract (FIG. 17, lanes 2-7, extract wasprepared as described above) were mixed together in a buffer containing10 mM Tris-Cl, pH 8.5, 50 mM KCl and 1.5 mM MgCl₂. The final reactionvolume was 10 μl. Reactions shown in lanes 4 and 7 contain in addition50 μM of each dNTP. Reactions shown in lanes 3, 4, 6 and 7 contain 0.2μM of the primer oligonucleotide (complementary to the 3′ arm of thesubstrate and shown in FIG. 16E). Reactions were incubated at 55° C. for4 minutes. Reactions were stopped by the addition of 8 μl of 95%formamide containing 20 mM EDTA and 0.05% marker dyes per 10 μl reactionvolume. Samples were then applied to 12% denaturing acrylamide gels.Following electrophoresis, the gels were autoradiographed. FIG. 17 showsthat clones 4E and 5B exhibit cleavage activity similar to that of thenative DNAPTaq. Note that some cleavage occurs in these reactions in theabsence of the primer. When long hairpin structure, such as the one usedhere (FIG. 16E), are used in cleavage reactions performed in bufferscontaining 50 mM KCl a low level of primer-independent cleavage is seen.Higher concentrations of KCl suppress, but do not eliminate, thisprimer-independent cleavage under these conditions.

[0413] 2. Assay for Synthetic Activity

[0414] The ability of the modified enzyme or proteolytic fragments isassayed by adding the modified enzyme to an assay system in which aprimer is annealed to a template and DNA synthesis is catalyzed by theadded enzyme. Many standard laboratory techniques employ such an assay.For example, nick translation and enzymatic sequencing involve extensionof a primer along a DNA template by a polymerase molecule.

[0415] In a preferred assay for determining the synthetic activity of amodified enzyme an oligonucleotide primer is annealed to asingle-stranded DNA template, e.g., bacteriophage M13 DNA, and theprimer/template duplex is incubated in the presence of the modifiedpolymerase in question, deoxynucleoside triphosphates (dNTPs) and thebuffer and salts known to be appropriate for the unmodified or nativeenzyme. Detection of either primer extension (by denaturing gelelectrophoresis) or dNTP incorporation (by acid precipitation orchromatography) is indicative of an active polymerase. A label, eitherisotopic or non-isotopic, is preferably included on either the primer oras a dNTP to facilitate detection of polymerization products. Syntheticactivity is quantified as the amount of free nucleotide incorporatedinto the growing DNA chain and is expressed as amount incorporated perunit of time under specific reaction conditions.

[0416] Representative results of an assay for synthetic activity isshown in FIG. 18. The synthetic activity of the mutant DNAPTaq clones4B-F was tested as follows: A master mixture of the following buffer wasmade: 1.2X PCR buffer (1× PCR buffer contains 50 mM KCl, 1.5 mM MgCl₂,10 mM Tris-Cl, ph 8.5 and 0.05% each Tween 20 and Nonidet P40), 50 μMeach of dGTP, dATP and dTTP, 5 μM dCTP and 0.125 μM α-³²P-dCTP at 600Ci/mmol. Before adjusting this mixture to its final volume, it wasdivided into two equal aliquots. One received distilled water up to avolume of 50 μl to give the concentrations above. The other received 5μg of single-stranded M13mp18 DNA (approximately 2.5 pmol or 0.05 μMfinal concentration) and 250 pmol of M13 sequencing primer (5 μM finalconcentration) and distilled water to a final volume of 50 μl. Eachcocktail was warmed to 75° C. for 5 minutes and then cooled to roomtemperature. This allowed the primers to anneal to the DNA in theDNA-containing mixtures.

[0417] For each assay, 4 μl of the cocktail with the DNA was combinedwith 1 μl of the mutant polymerase, prepared as described, or 1 unit ofDNAPTaq (Perkin Elmer) in 1 μl of dH₂O. A “no DNA” control was done inthe presence of the DNAPTaq (FIG. 18, lane 1), and a “no enzyme” controlwas done using water in place of the enzyme (lane 2). Each reaction wasmixed, then incubated at room temperature (approx. 22° C.) for 5minutes, then at 55° C. for 2 minutes, then at 72° C. for 2 minutes.

[0418] This step incubation was done to detect polymerization in anymutants that might have optimal temperatures lower than 72° C. After thefinal incubation, the tubes were spun briefly to collect anycondensation and were placed on ice. One μl of each reaction was spottedat an origin 1.5 cm from the bottom edge of a polyethyleneimine (PEI)cellulose thin layer chromatography plate and allowed to dry. Thechromatography plate was run in 0.75 M NaH₂PO₄, pH 3.5, until the bufferfront had run approximately 9 cm from the origin. The plate was dried,wrapped in plastic wrap, marked with luminescent ink, and exposed toX-ray film. Incorporation was detected as counts that stuck whereoriginally spotted, while the unincorporated nucleotides were carried bythe salt solution from the origin.

[0419] Comparison of the locations of the counts with the two controllanes confirmed the lack of polymerization activity in the mutantpreparations. Among the modified DNAPTaq clones, only clone 4B retainsany residual synthetic activity as shown in FIG. 18.

EXAMPLE 3 5′ Nucleases Derived from Thermostable DNA Polymerases canCleave Short Hairpin Structures with Specificity

[0420] The ability of the 5′ nucleases to cleave hairpin structures togenerate a cleaved hairpin structure suitable as a detection moleculewas examined. The structure and sequence of the hairpin test molecule isshown in FIG. 19A (SEQ ID NO:15). The oligonucleotide (labeled “primer”in FIG. 19A, SEQ ID NO:22) is shown annealed to its complementarysequence on the 3′ arm of the hairpin test molecule. The hairpin testmolecule was single-end labeled with ³²P using a labeled T7 promoterprimer in a polymerase chain reaction. The label is present on the 5′arm of the hairpin test molecule and is represented by the star in FIG.19A.

[0421] The cleavage reaction was performed by adding 10 fmoles ofheat-denatured, end-labeled hairpin test molecule, 0.2 uM of the primeroligonucleotide (complementary to the 3′ arm of the hairpin), 50 μM ofeach dNTP and 0.5 units of DNAPTaq (Perkin Elmer) or 0.5 μl of extractcontaining a 5′ nuclease (prepared as described above) in a total volumeof 10 μl in a buffer containing 10 mM Tris-Cl, pH 8.5, 50 mM KCl and 1.5mM MgCl₂. Reactions shown in lanes 3, 5 and 7 were run in the absence ofdNTPs.

[0422] Reactions were incubated at 55° C. for 4 minutes. Reactions werestopped at 55° C. by the addition of 8 μl of 95% formamide with 20 mMEDTA and 0.05% marker dyes per 10 μl reaction volume. Samples were notheated before loading onto denaturing polyacrylamide gels (10%polyacrylamide, 19:1 crosslinking, 7 M urea, 89 mM Tris-borate, pH 8.3,2.8 mM EDTA). The samples were not heated to allow for the resolution ofsingle-stranded and re-duplexed uncleaved hairpin molecules.

[0423]FIG. 19B shows that altered polymerases lacking any detectablesynthetic activity cleave a hairpin structure when an oligonucleotide isannealed to the single-stranded 3′ arm of the hairpin to yield a singlespecies of cleaved product (FIG. 19B, lanes 3 and 4). 5′ nucleases, suchas clone 4D, shown in lanes 3 and 4, produce a single cleaved producteven in the presence of dNTPs. 5′ nucleases which retain a residualamount of synthetic activity (less than 1% of wild type activity)produce multiple cleavage products as the polymerase can extend theoligonucleotide annealed to the 3′ arm of the hairpin thereby moving thesite of cleavage (clone 4B, lanes 5 and 6). Native DNATaq produces evenmore species of cleavage products than do mutant polymerases retainingresidual synthetic activity and additionally converts the hairpinstructure to a double-stranded form in the presence of dNTPs due to thehigh level of synthetic activity in the native polymerase (FIG. 19B,lane 8).

EXAMPLE 4 Test of the Trigger/Detection Assay

[0424] To test the ability of an oligonucleotide of the type released inthe trigger reaction of the trigger/detection assay to be detected inthe detection reaction of the assay, the two hairpin structures shown inFIG. 20A were synthesized using standard techniques. The two hairpinsare termed the A-hairpin (SEQ ID NO:23) and the T-hairpin (SEQ IDNO:24). The predicted sites of cleavage in the presence of theappropriate annealed primers are indicated by the arrows. The A- andT-hairpins were designed to prevent intra-strand mis-folding by omittingmost of the T residues in the A-hairpin and omitting most of the Aresidues in the T-hairpin. To avoid mis-priming and slippage, thehairpins were designed with local variations in the sequence motifs(e.g., spacing T residues one or two nucleotides apart or in pairs). TheA- and T-hairpins can be annealed together to form a duplex which hasappropriate ends for directional cloning in pUC-type vectors;restriction sites are located in the loop regions of the duplex and canbe used to elongate the stem regions if desired.

[0425] The sequence of the test trigger oligonucleotide is shown in FIG.20B; this oligonucleotide is termed the alpha primer (SEQ ID NO:25). Thealpha primer is complementary to the 3′ arm of the T-hairpin as shown inFIG. 20A. When the alpha primer is annealed to the T-hairpin, a cleavagestructure is formed that is recognized by thermostable DNA polymerases.Cleavage of the T-hairpin liberates the 5′ single-stranded arm of theT-hairpin, generating the tau primer (SEQ ID NO:26) and a cleavedT-hairpin (FIG. 20B; SEQ ID NO:27). The tau primer is complementary tothe 3′ arm of the A-hairpin as shown in FIG. 20A. Annealing of the tauprimer to the A-hairpin generates another cleavage structure; cleavageof this second cleavage structure liberates the 5′ single-stranded armof the A-hairpin, generating another molecule of the alpha primer whichthen is annealed to another molecule of the T-hairpin. Thermocyclingreleases the primers so they can function in additional cleavagereactions. Multiple cycles of annealing and cleavage are carried out.The products of the cleavage reactions are primers and the shortenedhairpin structures shown in FIG. 20C. The shortened or cleaved hairpinstructures may be resolved from the uncleaved hairpins byelectrophoresis on denaturing acrylamide gels.

[0426] The annealing and cleavage reactions are carried as follows: In a50 μl reaction volume containing 10 mM Tris-Cl, pH 8.5, 1.0 MgCl₂, 75 MMKCl, 1 pmole of A-hairpin, 1 pmole T-hairpin, the alpha primer is addedat equimolar amount relative to the hairpin structures (1 pmole) or atdilutions ranging from 10- to 10⁶-fold and 0.5 μl of extract containinga 5′ nuclease (prepared as described above) are added. The predictedmelting temperature for the alpha or trigger primer is 60° C. in theabove buffer. Annealing is performed just below this predicted meltingtemperature at 55° C. Using a Perkin Elmer DNA Thermal Cycler, thereactions are annealed at 55° C. for 30 seconds. The temperature is thenincreased slowly over a five minute period to 72° C. to allow forcleavage. After cleavage, the reactions are rapidly brought to 55° C.(1° C. per second) to allow another cycle of annealing to occur. A rangeof cycles are performed (20, 40 and 60 cycles) and the reaction productsare analyzed at each of these number of cycles. The number of cycleswhich indicates that the accumulation of cleaved hairpin products hasnot reached a plateau is then used for subsequent determinations when itis desirable to obtain a quantitative result.

[0427] Following the desired number of cycles, the reactions are stoppedat 55° C. by the addition of 8 μl of 95% formamide with 20 mM EDTA and0.05% marker dyes per 10 μl reaction volume. Samples are not heatedbefore loading onto denaturing polyacrylamide gels (10% polyacrylamide,19:1 crosslinking, 7 M urea, 89 mM tris-borate, pH 8.3, 2.8 mM EDTA).The samples were not heated to allow for the resolution ofsingle-stranded and re-duplexed uncleaved hairpin molecules.

[0428] The hairpin molecules may be attached to separate solid supportmolecules, such as agarose, styrene or magnetic beads, via the 3′ end ofeach hairpin. A spacer molecule may be placed between the 3′ end of thehairpin and the bead if so desired. The advantage of attaching thehairpins to a solid support is that this prevents the hybridization ofthe A- and T-hairpins to one another during the cycles of melting andannealing. The A- and T-hairpins are complementary to one another (asshown in FIG. 20D) and if allowed to anneal to one another over theirentire lengths this would reduce the amount of hairpins available forhybridization to the alpha and tau primers during the detectionreaction.

[0429] The 5′ nucleases of the present invention are used in this assaybecause they lack significant synthetic activity. The lack of syntheticactivity results in the production of a single cleaved hairpin product(as shown in FIG. 19B, lane 4). Multiple cleavage products may begenerated by 1) the presence of interfering synthetic activity (see FIG.19B, lanes 6 and 8) or 2) the presence of primer-independent cleavage inthe reaction. The presence of primer-independent cleavage is detected inthe trigger/detection assay by the presence of different sized productsat the fork of the cleavage structure. Primer-independent cleavage canbe dampened or repressed, when present, by the use of uncleavablenucleotides in the fork region of the hairpin molecule. For example,thiolated nucleotides can be used to replace several nucleotides at thefork region to prevent primer-independent cleavage.

EXAMPLE 5 Cleavage of Linear Nucleic Acid Substrates

[0430] From the above, it should be clear that native (i.e., “wildtype”) thermostable DNA polymerases are capable of cleaving hairpinstructures in a specific manner and that this discovery can be appliedwith success to a detection assay. In this example, the mutant DNAPs ofthe present invention are tested against three different cleavagestructures shown in FIG. 22A. Structure 1 in FIG. 22A is simply singlestranded 206-mer (the preparation and sequence information for which wasdiscussed above). Structures 2 and 3 are duplexes; structure 2 is thesame hairpin structure as shown in FIG. 12A (bottom), while structure 3has the hairpin portion of structure 2 removed.

[0431] The cleavage reactions comprised 0.01 pmoles of the resultingsubstrate DNA, and 1 pmole of pilot oligonucleotide in a total volume of10 μl of 10 MM Tris-Ci, pH 8.3, 100 mM KCl, 1 mM MgCl₂. Reactions wereincubated for 30 minutes at 55° C., and stopped by the addition of 8 μlof 95% formamide with 20 mM EDTA and 0.05% marker dyes. Samples wereheated to 75° C. for 2 minutes immediately before electrophoresisthrough a 10% polyacrylamide gel (19:1 cross link), with 7M urea, in abuffer of 45 mM Tris-Borate, pH 8.3, 1.4 mM EDTA.

[0432] The results were visualized by autoradiography and are shown inFIG. 22B with the enzymes indicated as follows: I is native Taq DNAP; IIis native Tfl DNAP; III is Cleavase® BX shown in FIG. 4E; IV isCleavase® BB shown in FIG. 4F; V is the mutant shown in FIG. 5B; and VIis Cleavase® BN shown in FIG. 4G.

[0433] Structure 2 was used to “normalize” the comparison. For example,it was found that it took 50 ng of Taq DNAP and 300 ng of Cleavase® BNto give similar amounts of cleavage of Structure 2 in thirty (30)minutes. Under these conditions native Taq DNAP is unable to cleaveStructure 3 to any significant degree. Native Tfl DNAP cleaves Structure3 in a manner that creates multiple products.

[0434] By contrast, all of the mutants tested cleave the linear duplexof Structure 3. This finding indicates that this characteristic of themutant DNA polymerases is consistent of thermostable polymerases acrossthermophilic species.

[0435] The finding described herein that the mutant DNA polymerases ofthe present invention are capable of cleaving linear duplex structuresallows for application to a more straightforward assay design (FIG. 1A).FIG. 23 provides a more detailed schematic corresponding to the assaydesign of FIG. 1A.

[0436] The two 43-mers depicted in FIG. 23 were synthesized by standardmethods. Each included a fluorescein on the 5′ for detection purposesand a biotin on the 3′ end to allow attachment to streptavidin coatedparamagnetic particles (the biotin-avidin attachment is indicated by“{circumflex over ()}”).

[0437] Before the trityl groups were removed, the oligos were purifiedby HPLC to remove truncated by-products of the synthesis reaction.Aliquots of each 43-mer were bound to M-280 Dynabeads (Dynal) at adensity of 100 pmoles per mg of beads. Two (2) mgs of beads (200 μl)were washed twice in 1× wash/bind buffer (1 M NaCl, 5 mM Tris-Cl, pH7.5, 0.5 mM EDTA) with 0.1% BSA, 200 μl per wash. The beads weremagnetically sedimented between washes to allow supernatant removal.After the second wash, the beads were resuspended in 200 μl of 2×wash/bind buffer (2 M Na Cl, 10 mM Tris-Cl, pH 7.5 with 1 mM EDTA), anddivided into two 100 μl aliquots. Each aliquot received 1 μl of a 100 μMsolution of one of the two oligonucleotides. After mixing, the beadswere incubated at room temperature for 60 minutes with occasional gentlemixing. The beads were then sedimented and analysis of the supernatantsshowed only trace amounts of unbound oligonucleotide, indicatingsuccessful binding. Each aliquot of beads was washed three times, 100 μlper wash, with IX wash/bind buffer, then twice in a buffer of 10 mMTris-Cl, pH 8.3 and 75 mM KCl. The beads were resuspended in a finalvolume of 100 μl of the Tris/KCl, for a concentration of 1 pmole ofoligo bound to 10 μg of beads per μl of suspension. The beads werestored at 4° C. between uses.

[0438] The types of beads correspond to FIG. 1A. That is to say, type 2beads contain the oligo (SEQ ID NO:33) comprising the complementarysequence (SEQ ID NO:34) for the alpha signal oligo (SEQ ID NO:35) aswell as the beta signal oligo (SEQ ID NO:36) which when liberated is a24-mer. This oligo has no “As” and is “T” rich. Type 3 beads contain theoligo (SEQ ID NO:37) comprising the complementary sequence (SEQ IDNO:38) for the beta signal oligo (SEQ ID NO:39) as well as the alphasignal oligo (SEQ ID NO:35) which when liberated is a 20-mer. This oligohas no “Ts” and is “A” rich.

[0439] Cleavage reactions comprised 1 μl of the indicated beads, 10pmoles of unlabelled alpha signal oligo as “pilot” (if indicated) and500 ng of Cleavase® BN in 20 μl of 75 mM KCl, 10 mM Tris-Cl, pH 8.3, 1.5mM MgCl₂ and 10 μM CTAB. All components except the enzyme wereassembled, overlaid with light mineral oil and warmed to 53° C. Thereactions were initiated by the addition of prewarmed enzyme andincubated at that temperature for 30 minutes. Reactions were stopped attemperature by the addition of 16 μl of 95% formamide with 20 mM EDTAand 0.05% each of bromophenol blue and xylene cyanol. This additionstops the enzyme activity and, upon heating, disrupts the biotin-avidinlink, releasing the majority (greater than 95%) of the oligos from thebeads. Samples were heated to 75° C. for 2 minutes immediately beforeelectrophoresis through a 10% polyacrylamide gel (19:1 cross link), with7 M urea, in a buffer of 45 mM Tris-Borate, pH 8.3, 1.4 mM EDTA. Resultswere visualized by contact transfer of the resolved DNA to positivelycharged nylon membrane and probing of the blocked membrane with ananti-fluorescein antibody conjugated to alkaline phosphatase. Afterwashing, the signal was developed by incubating the membrane in WesternBlue (Promega) which deposits a purple precipitate where the antibody isbound.

[0440]FIG. 24 shows the propagation of cleavage of the linear duplexnucleic acid structures of FIG. 23 by the DNAP mutants of the presentinvention. The two center lanes contain both types of beads. As notedabove, the beta signal oligo (SEQ ID NO:36) when liberated is a 24-merand the alpha signal oligo (SEQ ID NO:35) when liberated is a 20-mer.The formation of the two lower bands corresponding to the 24-mer and20-mer is clearly dependent on “pilot”.

EXAMPLE 6

[0441] 5′ Exonucleolytic Cleavage (“Nibbling”) by Thermostable DNAPs

[0442] It has been found that thermostable DNAPs, including those of thepresent invention, have a true 5′ exonuclease capable of nibbling the 5′end of a linear duplex nucleic acid structures. In this example, the 206base pair DNA duplex substrate is again employed (see above). In thiscase, it was produced by the use of one ³²P labeled primer and oneunlabeled primer in a polymerase chain reaction. The cleavage reactionscomprised 0.01 pmoles of heat-denatured, end-labeled substrate DNA (withthe unlabeled strand also present), 5 pmoles of pilot oligonucleotide(see pilot oligos in FIG. 12A) and 0.5 units of DNAPTaq or 0.5 μl ofCleavase® BB in the E. coli extract (see above), in a total volume of 10μl of 10 mM Tris-Cl, pH 8.5, 50 mM KCl, 1.5 mM MgCl₂.

[0443] Reactions were initiated at 65° C. by the addition of pre-warmedenzyme, then shifted to the final incubation temperature for 30 minutes.The results are shown in FIG. 25A. Samples in lanes 1-4 are the resultswith native Taq DNAP, while lanes 5-8 shown the results with Cleavase®BB. The reactions for lanes 1, 2, 5, and 6 were performed at 65° C. andreactions for lanes 3, 4, 7, and 8 were performed at 50° C. and all werestopped at temperature by the addition of 8 μl of 95% formamide with 20mM EDTA and 0.05% marker dyes. Samples were heated to 75° C. for 2minutes immediately before electrophoresis through a 10% acrylamide gel(19:1 cross-linked), with 7 M urea, in a buffer of 45 mM TrisBorate, pH8.3, 1.4 mM EDTA. The expected product in reactions 1, 2, 5, and 6 is 85nucleotides long; in reactions 3 and 7, the expected product is 27nucleotides long. Reactions 4 and 8 were performed without pilot, andshould remain at 206 nucleotides. The faint band seen at 24 nucleotidesis residual end-labeled primer from the PCR.

[0444] The surprising result is that Cleavase® BB under these conditionscauses all of the label to appear in a very small species, suggestingthe possibility that the enzyme completely hydrolyzed the substrate. Todetermine the composition of the fastest-migrating band seen in lanes5-8 (reactions performed with the deletion mutant), samples of the 206base pair duplex were treated with either T7 gene 6 exonuclease (USB) orwith calf intestine alkaline phosphatase (Promega), according tomanufacturers' instructions, to produce either labeled mononucleotide(lane a of FIG. 25B) or free ³²P-labeled inorganic phosphate (lane b ofFIG. 25B), respectively. These products, along with the products seen inlane 7 of panel A were resolved by brief electrophoresis through a 20%acrylamide gel (19:1 cross-link), with 7 M urea, in a buffer of 45 mMTris-Borate, pH 8.3, 1.4 mM EDTA. Cleavase® BB is thus capable ofconverting the substrate to mononucleotides.

EXAMPLE 7 Nibbling is Duplex Dependent

[0445] The nibbling by Cleavase® BB is duplex dependent. In thisexample, internally labeled, single strands of the 206-mer were producedby 15 cycles of primer extension incorporating α-³²P labeled dCTPcombined with all four unlabeled dNTPs, using an unlabeled 206-bpfragment as a template. Single and double stranded products wereresolved by electrophoresis through a non-denaturing 6% polyacrylamidegel (29:1 cross-link) in a buffer of 45 mM TrisBorate, pH 8.3, 1.4 mMEDTA, visualized by autoradiography, excised from the gel, eluted bypassive diffusion, and concentrated by ethanol precipitation.

[0446] The cleavage reactions comprised 0.04 pmoles of substrate DNA,and 2 μl of Cleavase® BB (in an E. coli extract as described above) in atotal volume of 40 μl of 10 mM Tris-Cl, pH 8.5, 50 mM KCl, 1.5 mM MgCl₂.Reactions were initiated by the addition of pre-warmed enzyme; 10 μlaliquots were removed at 5, 10, 20, and 30 minutes, and transferred toprepared tubes containing 8 μl of 95% formamide with 30 mM EDTA and0.05% marker dyes. Samples were heated to 75° C. for 2 minutesimmediately before electrophoresis through a 10% acrylamide gel (19:1cross-linked), with 7 M urea, in a buffer of 45 mM Tris-Borate, pH 8.3,1.4 mM EDTA. Results were visualized by autoradiography as shown in FIG.26. Clearly, the cleavage by Cleavase® BB depends on a duplex structure;no cleavage of the single strand structure is detected whereas cleavageof the 206-mer duplex is complete.

EXAMPLE 8 Nibbling can be Target Directed

[0447] The nibbling activity of the DNAPs of the present invention canbe employed with success in a detection assay. One embodiment of such anassay is shown in FIG. 27. In this assay, a labelled oligo is employedthat is specific for a target sequence. The oligo is in excess of thetarget so that hybridization is rapid. In this embodiment, the oligocontains two fluorescein labels whose proximity on the oligo causestheir emission to be quenched. When the DNAP is permitted to nibble theoligo the labels separate and are detectable. The shortened duplex isdestabilized and disassociates. Importantly, the target is now free toreact with an intact labelled oligo.

[0448] The reaction can continue until the desired level of detection isachieved. An analogous, although different, type of cycling assay hasbeen described employing lambda exonuclease. See C. G. Copley and C.Boot, BioTechniques 13:888 (1992).

[0449] The success of such an assay depends on specificity. In otherwords, the oligo must hybridize to the specific target. It is alsopreferred that the assay be sensitive; the oligo ideally should be ableto detect small amounts of target. FIG. 28A shows a 5′-end ³²P-labelledprimer bound to a plasmid target sequence. In this case, the plasmid waspUC19 (commercially available) which was heat denatured by boiling two(2) minutes and then quick chilling. The primer is a 21-mer (SEQ IDNO:39). The enzyme employed was Cleavase® BX (a dilution equivalent to5×10-3 μl extract) in 100 mM KCl, 10 mM Tris-Cl, pH 8.3, 2 mM MnCl₂. Thereaction was performed at 55° C. for sixteen (16) hours with or withoutgenomic background DNA (from chicken blood). The reaction was stopped bythe addition of 8 μl of 95% formamide with 20 mM EDTA and marker dyes.

[0450] The products of the reaction were resolved by PAGE (10%polyacrylamide, 19:1 cross link, 1× TBE) as seen in FIG. 28B. Lane “M”contains the labelled 21-mer. Lanes 1-3 contain no specific target,although Lanes 2 and 3 contain 100 ng and 200 ng of genomic DNA,respectively. Lanes 4, 5 and 6 all contain specific target with either 0ng, 100 ng or 200 ng of genomic DNA, respectively. It is clear thatconversion to mononucleotides occurs in Lanes 4, 5 and 6 regardless ofthe presence or amount of background DNA. Thus, the nibbling can betarget directed and specific.

EXAMPLE 9 Cleavage Purification

[0451] As noted above, expressed thermostable proteins, i.e., the 5′nucleases, were isolated by crude bacterial cell extracts. Theprecipitated E. coli proteins were then, along with other cell debris,removed by centrifugation. In this example, cells expressing the BNclone were cultured and collected (500 grams). For each gram (wetweight) of E. coli, 3 ml of lysis buffer (50 mM Tris-HCl, pH 8.0, 1 mMEDTA, 100 μM NaCl) was added. The cells were lysed with 200 μg/mllysozyme at room temperature for 20 minutes. Thereafter deoxycholic acidwas added to make a 0.2% final concentration and the mixture wasincubated 15 minutes at room temperature.

[0452] The lysate was sonicated for approximately 6-8 minutes at 0° C.The precipitate was removed by centrifugation (39,000 g for 20 minutes).Polyethyleneimine was added (0.5%) to the supernatant and the mixturewas incubated on ice for 15 minutes. The mixture was centrifuged (5,000g for 15 minutes) and the supernatant was retained. This was heated for30 minutes at 60° C. and then centrifuged again (5,000 g for 15 minutes)and the supernatant was again retained.

[0453] The supernatant was precipitated with 35% ammonium sulfate at 4°C. for 15 minutes. The mixture was then centrifuged (5,000 g for 15minutes) and the supernatant was removed. The precipitate was thendissolved in 0.25 M KCl, 20 Tris pH 7.6, 0.2% Tween and 0.1 EDTA) andthen dialyzed against Binding Buffer (8× Binding Buffer comprises: 40 mMimidazole, 4M NaCl, 160 mM Tris-HCl, pH 7.9).

[0454] The solubilized protein is then purified on the Ni⁺⁺ column(Novagen). The Binding Buffer is allows to drain to the top of thecolumn bed and load the column with the prepared extract. A flow rate ofabout 10 column volumes per hour is optimal for efficient purification.If the flow rate is too fast, more impurities will contaminate theeluted fraction.

[0455] The column is washed with 25 ml (10 volumes) of 1× Binding Bufferand then washed with 15 ml (6 volumes) of 1× Wash Buffer (8× Wash Buffercomprises: 480 mM imidazole, 4M NaCl, 160 mM Tris-HCl, pH 7.9). Thebound protein was eluted with 15 ml (6 volumes) of 1× Elute Buffer (4×Elute Buffer comprises: 4 mM imidazole, 2 M NaCl, 80 mM Tris-HCl, pH7.9). Protein is then reprecipitated with 35% Ammonium Sulfate as above.The precipitate was then dissolved and dialyzed against: 20 mM Tris, 100mM KCl, 1 mM EDTA). The solution was brought up to 0.1% each of Tween 20and NP-40 and stored at 4° C.

EXAMPLE 10 The Use of Various Divalent Cations in the Cleavage ReactionInfluences the Nature of the Resulting Cleavage Products

[0456] In comparing the 5′ nucleases generated by the modificationand/or deletion of the C-terminal polymerization domain of Thermusaquaticus DNA polymerase (DNAPTaq), as diagrammed in FIGS. 4B-G,significant differences in the strength of the interactions of theseproteins with the 3′ end of primers located upstream of the cleavagesite (as depicted in FIG. 6) were noted. In describing the cleavage ofthese structures by Pol I-type DNA polymerases [Example 1 and Lyamichevet al. (1993) Science 260:778], it was observed that in the absence of aprimer, the location of the junction between the double-stranded regionand the single-stranded 5′ and 3′ arms determined the site of cleavage,but in the presence of a primer, the location of the 3′ end of theprimer became the determining factor for the site of cleavage. It waspostulated that this affinity for the 3′ end was in accord with thesynthesizing function of the DNA polymerase.

[0457] Structure 2, shown in FIG. 22A, was used to test the effects of a3′ end proximal to the cleavage site in cleavage reactions comprisingseveral different solutions [e.g., solutions containing different salts(KCl or NaCl), different divalent cations (Mn²⁺ or Mg²⁺), etc.] as wellas the use of different temperatures for the cleavage reaction. When thereaction conditions were such that the binding of the enzyme (e.g., aDNAP comprising a 5′ nuclease, a modified DNAP or a 5′ nuclease) to the3′ end (of the pilot oligonucleotide) near the cleavage site was strong,the structure shown is cleaved at the site indicated in FIG. 22A. Thiscleavage releases the unpaired 5′ arm and leaves a nick between theremaining portion of the target nucleic acid and the folded 3′ end ofthe pilot oligonucleotide. In contrast, when the reaction conditions aresuch that the binding of the DNAP (comprising a 5′ nuclease) to the 3′end was weak, the initial cleavage was as described above, but after therelease of the 5′ arm, the remaining duplex is digested by theexonuclease function of the DNAP.

[0458] One way of weakening the binding of the DNAP to the 3′ end is toremove all or part of the domain to which at least some of this functionhas been attributed. Some of 5′ nucleases created by deletion of thepolymerization domain of DNAPTaq have enhanced true exonucleasefunction, as demonstrated in Example 6.

[0459] The affinity of these types of enzymes (i.e., 5′ nucleasesassociated with or derived from DNAPs) for recessed 3′ ends may also beaffected by the identity of the divalent cation present in the cleavagereaction. It was demonstrated by Longley et al. [Nucl. Acids Res.18:7317 (1990)] that the use of MnCl₂ in a reaction with DNAPTaq enabledthe polymerase to remove nucleotides from the 5′ end of a primerannealed to a template, albeit inefficiently. Similarly, by examinationof the cleavage products generated using Structure 2 from FIG. 22A, asdescribed above, in a reaction containing either DNAPTaq or theCleavase® BB nuclease, it was observed that the substitution of MnCl₂for MgCl₂ in the cleavage reaction resulted in the exonucleolytic“nibbling” of the duplex downstream of the initial cleavage site. Whilenot limiting the invention to any particular mechanism, it is thoughtthat the substitution of MnCl₂ for MgCl₂ in the cleavage reactionlessens the affinity of these enzymes for recessed 3′ ends.

[0460] In all cases, the use of MnCl₂ enhances the 5′ nuclease function,and in the case of the Cleavase® BB nuclease, a 50- to 100-foldstimulation of the 5′ nuclease function is seen. Thus, while theexonuclease activity of these enzymes was demonstrated above in thepresence of MgCl₂, the assays described below show a comparable amountof exonuclease activity using 50 to 100-fold less enzyme when MnCl₂ isused in place of MgCl₂. When these reduced amounts of enzyme are used ina reaction mixture containing MgCl₂, the nibbling or exonucleaseactivity is much less apparent than that seen in Examples 6-8.

[0461] Similar effects are observed in the performance of the nucleicacid detection assay described in Examples 11-18 below when reactionsperformed in the presence of either MgCl₂ or MnCl₂ are compared. In thepresence of either divalent cation, the presence of the invaderoligonucleotide (described below) forces the site of cleavage into theprobe duplex, but in the presence of MnCl₂ the probe duplex can befurther nibbled producing a ladder of products that are visible when a3′ end label is present on the probe oligonucleotide. When the invaderoligonucleotide is omitted from a reaction containing Mn²⁺, the probe isnibbled from the 5′ end. Mg²⁺-based reactions display minimal nibblingof the probe oligonucleotide. In any of these cases, the digestion ofthe probe is dependent upon the presence of the target nucleic acid. Inthe examples below, the ladder produced by the enhanced nibblingactivity observed in the presence of Mn²⁺ is used as a positiveindicator that the probe oligonucleotide has hybridized to the targetsequence.

EXAMPLE 11 Invasive 5′ Endonucleolytic Cleavage by Thermostable 5′Nucleases in the Absence of Polymerization

[0462] As described in the examples above, 5′ nucleases cleave near thejunction between single-stranded and base-paired regions in a bifurcatedduplex, usually about one base pair into the base-paired region. In thisexample, it is shown that thermostable 5′ nucleases, including those ofthe present invention (e.g., Cleavase® BN nuclease, Cleavase® A/Gnuclease), have the ability to cleave a greater distance into the basepaired region when provided with an upstream oligonucleotide bearing a3′ region that is homologous to a 5′ region of the subject duplex, asshown in FIG. 30.

[0463]FIG. 30 shows a synthetic oligonucleotide which was designed tofold upon itself which consists of the following sequence:5′-GTTCTCTGCTCTCTGGTCGCTG TCTCGCTTGTGAAACAAGCGAGACAGCGTGGTCTCTCG-3′ (SEQID NO:40). This oligonucleotide is referred to as the “S-60 Hairpin.”The 15 basepair hairpin formed by this oligonucleotide is furtherstabilized by a “tri-loop” sequence in the loop end (i.e., threenucleotides form the loop portion of the hairpin) [Hiraro, I. et al.(1994) Nucleic Acids Res. 22(4):576]. FIG. 30 also show the sequence ofthe P-15 oligonucleotide and the location of the region ofcomplementarity shared by the P-15 and S-60 hairpin oligonucleotides.The sequence of the P-15 oligonucleotide is 5′-CGAGAGACCACGCTG-3′ (SEQID NO:41). As discussed in detail below, the solid black arrowheadsshown in FIG. 29 indicate the sites of cleavage of the S-60 hairpin inthe absence of the P-15 oligonucleotide and the hollow arrow headsindicate the sites of cleavage in the presence of the P-15oligonucleotide. The size of the arrow head indicates the relativeutilization of a particular site.

[0464] The S-60 hairpin molecule was labeled on its 5′ end with biotinfor subsequent detection. The S-60 hairpin was incubated in the presenceof a thermostable 5′ nuclease in the presence or the absence of the P-15oligonucleotide. The presence of the full duplex which can be formed bythe S-60 hairpin is demonstrated by cleavage with the Cleavage® BN 5′nuclease, in a primer-independent fashion (i.e., in the absence of theP-15 oligonucleotide). The release of 18 and 19-nucleotide fragmentsfrom the 5′ end of the S-60 hairpin molecule showed that the cleavageoccurred near the junction between the single and double strandedregions when nothing is hybridized to the 3′ arm of the S-60 hairpin(FIG. 31, lane 2).

[0465] The reactions shown in FIG. 31 were conducted as follows. Twentyfmole of the 5′ biotin-labeled hairpin DNA (SEQ ID NO:40) was combinedwith 0.1 ng of Cleavase® BN enzyme and 1 μl of 100 mM MOPS (pH 7.5)containing 0.5% each of Tween-20 and NP-40 in a total volume of 9 μl. Inthe reaction shown in lane 1, the enzyme was omitted and the volume wasmade up by addition of distilled water (this served as the uncut or noenzyme control). The reaction shown in lane 3 of FIG. 31 also included0.5 pmole of the P15 oligonucleotide (SEQ ID NO:41), which can hybridizeto the unpaired 3′ arm of the S-60 hairpin (SEQ ID NO:40), as diagrammedin FIG. 30.

[0466] The reactions were overlaid with a drop of mineral oil, heated to95° C. for 15 seconds, then cooled to 37° C., and the reaction wasstarted by the addition of 1 μl of 10 mM MnCl₂ to each tube. After 5minutes, the reactions were stopped by the addition of 6 μl of 95%formamide containing 20 mM EDTA and 0.05% marker dyes. Samples wereheated to 75° C. for 2 minutes immediately before electrophoresisthrough a 15% acrylamide gel (19:1 cross-linked), with 7 M urea, in abuffer of 45 mM Tris-Borate, pH 8.3, 1.4 mM EDTA.

[0467] After electrophoresis, the gel plates were separated allowing thegel to remain flat on one plate. A 0.2 mm-pore positively-charged nylonmembrane (NYTRAN, Schleicher and Schuell, Keene, N.H.), pre-wetted inH₂O, was laid on top of the exposed gel. All air bubbles were removed.Two pieces of 3MM filter paper (Whatman) were then placed on top of themembrane, the other glass plate was replaced, and the sandwich wasclamped with binder clips. Transfer was allowed to proceed overnight.After transfer, the membrane was carefully peeled from the gel andallowed to air dry. After complete drying, the membrane was washed in1.2× Sequenase Images Blocking Buffer (United States Biochemical) using0.3 ml of buffer/cm² of membrane. The wash was performed for 30 minutesat room temperature. A streptavidin-alkaline phosphatase conjugate(SAAP, United States Biochemical) was added to a 1:4000 dilutiondirectly to the blocking solution, and agitated for 15 minutes. Themembrane was rinsed briefly with H20 and then washed three times for 5minutes per wash using 0.5 ml/cm² of 1× SAAP buffer (100 mM Tris-HCl, pH10, 50 mM NaCl) with 0.1% sodium dodecyl sulfate (SDS). The membrane wasrinsed briefly with H20 between each wash. The membrane was then washedonce in 1× SAAP buffer containing 1 MM MgCl₂ without SDS, drainedthoroughly and placed in a plastic heat-sealable bag. Using a sterilepipet, 5 mls of CDP-Starm (Tropix, Bedford, Mass.) chemiluminescentsubstrate for alkaline phosphatase were added to the bag and distributedover the entire membrane for 2-3 minutes. The CDP-Star™-treated membranewas exposed to XRP X-ray film (Kodak) for an initial exposure of 10minutes.

[0468] The resulting autoradiograph is shown in FIG. 31. In FIG. 31, thelane labelled “M” contains the biotinylated P-15 oligonucleotide whichserved as a marker. The sizes (in nucleotides) of the uncleaved S-60hairpin (60 nuc; lane 1), the marker (15 nuc; lane “M”) and the cleavageproducts generated by cleavage of the S-60 hairpin in the presence (lane3) or absence (lane 2) of the P-15 oligonucleotide are indicated.

[0469] Because the complementary regions of the S-60 hairpin are locatedon the same molecule, essentially no lag time should be needed to allowhybridization (i.e., to form the duplex region of the hairpin). Thishairpin structure would be expected to form long before the enzyme couldlocate and cleave the molecule. As expected, cleavage in the absence ofthe primer oligonucleotide was at or near the junction between theduplex and single-stranded regions, releasing the unpaired 5′ arm (FIG.31, lane 2). The resulting cleavage products were 18 and 19 nucleotidesin length.

[0470] It was expected that stability of the S-60 hairpin with thetri-loop would prevent the P-15 oligonucleotide from promoting cleavagein the “primer-directed” manner described in Example 1 above, becausethe 3′ end of the “primer” would remain unpaired. Surprisingly, it wasfound that the enzyme seemed to mediate an “invasion” by the P-15 primerinto the duplex region of the S-60 hairpin, as evidenced by the shiftingof the cleavage site 3 to 4 basepairs further into the duplex region,releasing the larger products (22 and 21 nuc.) observed in lane 3 ofFIG. 31.

[0471] The precise sites of cleavage of the S-60 hairpin are diagrammedon the structure in FIG. 30, with the solid black arrowheads indicatingthe sites of cleavage in the absence of the P-15 oligonucleotide and thehollow arrow heads indicating the sites of cleavage in the presence ofP-15.

[0472] These data show that the presence on the 3′ arm of anoligonucleotide having some sequence homology with the first severalbases of the similarly oriented strand of the downstream duplex can be adominant factor in determining the site of cleavage by 5′ nucleases.Because the oligonucleotide which shares some sequence homology with thefirst several bases of the similarly oriented strand of the downstreamduplex appears to invade the duplex region of the hairpin, it isreferred to as an “invader” oligonucleotide. As shown in the examplesbelow, an invader oligonucleotide appears to invade (or displace) aregion of duplexed nucleic acid regardless of whether the duplex regionis present on the same molecule (i.e., a hairpin) or whether the duplexis formed between two separate nucleic acid strands.

EXAMPLE 12 The Invader Oligonucleotide Shifts the Site of Cleavage in aPre-Formed Probe/Target Duplex

[0473] In Example 11 it was demonstrated that an invader oligonucleotidecould shift the site at which a 5′ nuclease cleaves a duplex regionpresent on a hairpin molecule. In this example, the ability of aninvader oligonucleotide to shift the site of cleavage within a duplexregion formed between two separate strands of nucleic acid molecules wasexamined.

[0474] A single-stranded target DNA comprising the single-strandedcircular M13mp19 molecule and a labeled (fluorescein) probeoligonucleotide were mixed in the presence of the reaction buffercontaining salt (KCl) and divalent cations (Mg²⁺ or Mn²⁺) to promoteduplex formation. The probe oligonucleotide refers to a labelledoligonucleotide which is complementary to a region along the targetmolecule (e.g., M13mp19). A second oligonucleotide (unlabelled) wasadded to the reaction after the probe and target had been allowed toanneal. The second oligonucleotide binds to a region of the target whichis located downstream of the region to which the probe oligonucleotidebinds. This second oligonucleotide contains sequences which arecomplementary to a second region of the target molecule. If the secondoligonucleotide contains a region which is complementary to a portion ofthe sequences along the target to which the probe oligonucleotide alsobinds, this second oligonucleotide is referred to as an invaderoligonucleotide (see FIG. 32c).

[0475]FIG. 32 depicts the annealing of two oligonucleotides to regionsalong the M13mp19 target molecule (bottom strand in all three structuresshown). In FIG. 32 only a 52 nucleotide portion of the M13mp19 moleculeis shown; this 52 nucleotide sequence is listed in SEQ ID NO:42. Theprobe oligonucleotide contains a fluorescein label at the 3′ end; thesequence of the probe is 5′-AGAAAGGAAGGGAAGAAAGC GAAAGG-3′ (SEQ IDNO:43). In FIG. 32, sequences comprising the second oligonucleotide,including the invader oligonucleotide are underlined. In FIG. 32a, thesecond oligonucleotide, which has the sequence 5′-GACGGGGAAAGCCGGCGAACG-3′ (SEQ ID NO:44), is complementary to a different and downstreamregion of the target molecule than is the probe oligonucleotide (labeledwith fluorescein or “Fluor”); there is a gap between the second,upstream oligonucleotide and the probe for the structure shown in FIG.32a. In FIG. 32b, the second, upstream oligonucleotide, which has thesequence 5′-GAAAGCCGGCGAACGTGGCG-3′ (SEQ ID NO:45), is complementary toa different region of the target molecule than is the probeoligonucleotide, but in this case, the second oligonucleotide and theprobe oligonucleotide abut one another (that is the 3′ end of thesecond, upstream oligonucleotide is immediately adjacent to the 5′ endof the probe such that no gap exists between these twooligonucleotides). In FIG. 32c, the second, upstream oligonucleotide[5′-GGCGAACGTGGCGAGAAAGGA-3′ (SEQ ID NO:46)] and the probeoligonucleotide share a region of complementarity with the targetmolecule. Thus, the upstream oligonucleotide has a 3′ arm which has asequence identical to the first several bases of the downstream probe.In this situation, the upstream oligonucleotide is referred to as an“invader” oligonucleotide.

[0476] The effect of the presence of an invader oligonucleotide upon thepattern of cleavage in a probe/target duplex formed prior to theaddition of the invader was examined. The invader oligonucleotide andthe enzyme were added after the probe was allowed to anneal to thetarget and the position and extent of cleavage of the probe wereexamined to determine a) if the invader was able to shift the cleavagesite to a specific internal region of the probe, and b), if the reactioncould accumulate specific cleavage products over time, even in theabsence of thermal cycling, polymerization, or exonuclease removal ofthe probe sequence.

[0477] The reactions were carried out as follows. Twenty μl each of twoenzyme mixtures were prepared, containing 2 μl of Cleavase® A/G nucleaseextract (prepared as described in Example 2), with or without 50 pmoleof the invader oligonucleotide (SEQ ID NO:46), as indicated, per 4 μl ofthe mixture. For each of the eight reactions shown in FIG. 33, 150 fmoleof M13mp19 single-stranded DNA (available from Life Technologies, Inc.)was combined with 5 pmoles of fluorescein labeled probe (SEQ ID NO:43),to create the structure shown in FIG. 31c, but without the invaderoligonucleotide present (the probe/target mixture). One half (4 tubes)of the probe/target mixtures were combined with 1 μl of 100 mM MOPS, pH7.5 with 0.5% each of Tween-20 and NP-40, 0.5 μl of 1 M KCl and 0.25 μlof 80 mM MnCl₂, and distilled water to a volume of 6 μl. The second setof probe/target mixtures were combined with 1 μl of 100 mM MOPS, pH 7.5with 0.5% each of Tween-20 and NP-40, 0.5 μl of 1 M KCl and 0.25 μl of80 mM MgCl₂. The second set of mixtures therefore contained MgCl₂ inplace of the MnCl₂ present in the first set of mixtures.

[0478] The mixtures (containing the probe/target with buffer, KCl anddivalent cation) were covered with a drop of ChillOut® evaporationbarrier (MJ Research) and were brought to 60° C. for 5 minutes to allowannealing. Four μl of the above enzyme mixtures without the invaderoligonucleotide was added to reactions whose products are shown in lanes1, 3, 5 and 7 of FIG. 33. Reactions whose products are shown lanes 2, 4,6, and 8 of FIG. 33 received the same amount of enzyme mixed with theinvader oligonucleotide (SEQ ID NO:46). Reactions 1, 2, 5 and 6 wereincubated for 5 minutes at 60° C. and reactions 3, 4, 7 and 8 wereincubated for 15 minutes at 60° C.

[0479] All reactions were stopped by the addition of 8 μl of 95%formamide with 20 mM EDTA and 0.05% marker dyes. Samples were heated to90° C. for 1 minute immediately before electrophoresis through a 20%acrylamide gel (19:1 cross-linked), containing 7 M urea, in a buffer of45 mM Tris-Borate, pH 8.3, 1.4 mM EDTA. Following electrophoresis, thereaction products and were visualized by the use of an Hitachi FMBIOfluorescence imager, the output of which is seen in FIG. 33. The verylow molecular weight fluorescent material seen in all lanes at or nearthe salt front in FIG. 33 and other fluoro-imager figures is observedwhen fluorescently-labeled oligonucleotides are electrophoresed andimaged on a fluoro-imager. This material is not a product of thecleavage reaction.

[0480] The use of MnCl₂ in these reactions (lanes 1-4) stimulates thetrue exonuclease or “nibbling” activity of the Cleavase® enzyme, asdescribed in Example 7, as is clearly seen in lanes 1 and 3 of FIG. 33.This nibbling of the probe oligonucleotide (SEQ ID NO:43) in the absenceof invader oligonucleotide (SEQ ID NO:46) confirms that the probeoligonucleotide is forming a duplex with the target sequence. Theladder-like products produced by this nibbling reaction may be difficultto differentiate from degradation of the probe by nucleases that mightbe present in a clinical specimen. In contrast, introduction of theinvader oligonucleotide (SEQ ID NO:46) caused a distinctive shift in thecleavage of the probe, pushing the site of cleavage 6 to 7 bases intothe probe, confirming the annealing of both oligonucleotides. Inpresence of MnCl₂, the exonuclease “nibbling” may occur after theinvader-directed cleavage event, until the residual duplex isdestabilized and falls apart.

[0481] In a magnesium based cleavage reaction (lanes 5-8), the nibblingor true exonuclease function of the Cleavase® A/G is enzyme suppressed(but the endonucleolytic function of the enzyme is essentiallyunaltered), so the probe oligonucleotide is not degraded in the absenceof the invader (FIG. 33, lanes 5 and 7). When the invader is added, itis clear that the invader oligonucleotide can promote a shift in thesite of the endonucleolytic cleavage of the annealed probe. Comparisonof the products of the 5 and 15 minute reactions with invader (lanes 6and 8 in FIG. 33) shows that additional probe hybridizes to the targetand is cleaved. The calculated melting temperature (Tm) of the portionof probe that is not invaded (i.e., nucleotides 9-26 of SEQ ID NO:43) is56° C., so the observed turnover (as evidenced by the accumulation ofcleavage products with increasing reaction time) suggests that the fulllength of the probe molecule, with a calculated Tm of 76° C., is must beinvolved in the subsequent probe annealing events in this 60° C.reaction.

EXAMPLE 13 The Overlap of the 3′ Invader Oligonucleotide Sequence withthe 5′ Region of the Probe Causes a Shift in the Site of Cleavage

[0482] In Example 12, the ability of an invader oligonucleotide to causea shift in the site of cleavage of a probe annealed to a target moleculewas demonstrated. In this example, experiments were conducted to examinewhether the presence of an oligonucleotide upstream from the probe wassufficient to cause a shift in the cleavage site(s) along the probe orwhether the presence of nucleotides on the 3′ end of the invaderoligonucleotide which have the same sequence as the first severalnucleotides at the 5′ end of the probe oligonucleotide were required topromote the shift in cleavage.

[0483] To examine this point, the products of cleavage obtained fromthree different arrangements of target-specific oligonucleotides arecompared. A diagram of these oligonucleotides and the way in which theyhybridize to a test nucleic acid, M13mp19, is shown in FIG. 32. In FIG.32a, the 3′ end of the upstream oligonucleotide (SEQ ID NO:45) islocated upstream of the 5′ end of the downstream “probe” oligonucleotide(SEQ ID NO:43) such that a region of the M13 target which is not pairedto either oligonucleotide is present. In FIG. 32b, the sequence of theupstream oligonucleotide (SEQ ID NO:45) is immediately upstream of theprobe (SEQ ID NO:43), having neither a gap nor an overlap between thesequences. FIG. 32c diagrams the arrangement of the substrates used inthe assay of the present invention, showing that the upstream “invader”oligonucleotide (SEQ ID NO:46) has the same sequence on a portion of its3′ region as that present in the 5′ region of the downstream probe (SEQID NO:43). That is to say, these regions will compete to hybridize tothe same segment of the M13 target nucleic acid.

[0484] In these experiments, four enzyme mixtures were prepared asfollows (planning 5 μl per digest): Mixture 1 contained 2.25 μl ofCleavase® A/G nuclease extract (prepared as described in Example 2) per5 μl of mixture, in 20 mM MOPS, pH 7.5 with 0.1% each of Tween 20 andNP-40, 4 mM MnCl₂ and 100 mM KCl. Mixture 2 contained 11.25 units of TaqDNA polymerase (Promega Corp., Madison, WI) per 5 μl of mixture in 20 mMMOPS, pH 7.5 with 0.1% each of Tween 20 and NP-40, 4 mM MnCl₂ and 100 mMKCl. Mixture 3 contained 2.25 μl of Cleavase® A/G nuclease extract per 5μl of mixture in 20 mM Tris-HCl, pH 8.5, 4 mM MgCl₂ and 100 mM KCl.Mixture 4 contained 11.25 units of Taq DNA polymerase per 5 μl ofmixture in 20 mM Tris-HCl, pH 8.5, 4 mM MgCl₂ and 100 mM KCl.

[0485] For each reaction, 50 fmole of M13mp19 single-stranded DNA (thetarget nucleic acid) was combined with 5 pmole of the probeoligonucleotide (SEQ ID NO:43 which contained a fluorescein label at the3′ end) and 50 pmole of one of the three upstream oligonucleotidesdiagrammed in FIG. 32 (i.e., one of SEQ ID NOS:44-46), in a total volumeof 5 μl of distilled water. The reactions were overlaid with a drop ofChillOut™ evaporation barrier (MJ Research) and warmed to 62° C. Thecleavage reactions were started by the addition of 5 μl of an enzymemixture to each tube, and the reactions were incubated at 62° C. for 30min. The reactions shown in lanes 1-3 of FIG. 34 received Mixture 1;reactions 4-6 received Mixture 2; reactions 7-9 received Mixture 3 andreactions 10-12 received Mixture 4.

[0486] After 30 minutes at 62° C., the reactions were stopped by theaddition of 8 μl of 95% formamide with 20 mM EDTA and 0.05% marker dyes.Samples were heated to 75° C. for 2 minutes immediately beforeelectrophoresis through a 20% acrylamide gel (19:1 cross-linked), with 7M urea, in a buffer of 45 mM Tris-Borate, pH 8.3, 1.4 mM EDTA.

[0487] Following electrophoresis, the products of the reactions werevisualized by the use of an Hitachi FMBIO fluorescence imager, theoutput of which is seen in FIG. 34. The reaction products shown in lanes1, 4, 7 and 10 of FIG. 34 were from reactions which contained SEQ IDNO:44 as the upstream oligonucleotide (see FIG. 32a). The reactionproducts shown in lanes 2, 5, 8 and 11 of FIG. 34 were from reactionswhich contained SEQ ID NO:45 as the upstream oligonucleotide (see FIG.32b). The reaction products shown in lanes 3, 6, 9 and 12 of FIG. 34were from reactions which contained SEQ ID NO:46, the invaderoligonucleotide, as the upstream oligonucleotide (see FIG. 32c).

[0488] Examination of the Mn²⁺ based reactions using either Cleavase®A/G nuclease or DNAPTaq as the cleavage agent (lanes 1 through 3 and 4through 6, respectively) shows that both enzymes have active exonucleasefunction in these buffer conditions. The use of a 3′ label on the probeoligonucleotide allows the products of the nibbling activity to remainlabeled, and therefore visible in this assay. The ladders seen in lanes1, 2, 4 and 5 confirm that the probe hybridize to the target DNA asintended. These lanes also show that the location of the non-invasiveoligonucleotides have little effect on the products generated. Theuniform ladder created by these digests would be difficult todistinguish from a ladder causes by a contaminating nuclease, as onemight find in a clinical specimen. In contrast, the products displayedin lanes 3 and 6, where an invader oligonucleotide was provided todirect the cleavage, show a very distinctive shift, so that the primarycleavage product is smaller than those seen in the non-invasivecleavage. This product is then subject to further nibbling in theseconditions, as indicated by the shorter products in these lanes. Theseinvader-directed cleavage products would be easily distinguished from abackground of non-specific degradation of the probe oligonucleotide.

[0489] When Mg²⁺ is used as the divalent cation the results are evenmore distinctive. In lanes 7, 8, 10 and 11 of FIG. 34, where theupstream oligonucleotides were not invasive, minimal nibbling isobserved. The products in the DNAPTaq reactions show some accumulationof probe that has been shortened on the 5′ end by one or two nucleotidesconsistent with previous examination of the action of this enzyme onnicked substrates (Longley et al., supra). When the upstreamoligonucleotide is invasive, however, the appearance of thedistinctively shifted probe band is seen. These data clearly indicatedthat it is the invasive 3′ portion of the upstream oligonucleotide thatis responsible for fixing the site of cleavage of the downstream probe.

[0490] Thus, the above results demonstrate that it is the presence ofthe free or initially non-annealed nucleotides at the 3′ end of theinvader oligonucleotide which mediate the shift in the cleavage site,not just the presence of an oligonucleotide annealed upstream of theprobe. Nucleic acid detection assays which employ the use of an invaderoligonucleotide are termed “invader-directed cleavage” assays.

EXAMPLE 14 Invader-Directed Cleavage Recognizes Single and DoubleStranded Target Molecules in a Background of Non-Target DNA Molecules

[0491] For a nucleic acid detection method to be broadly useful, it mustbe able to detect a specific target in a sample that may contain largeamounts of other DNA, e.g., bacterial or human chromosomal DNA. Theability of the invader directed cleavage assay to recognize and cleaveeither single- or double-stranded target molecules in the presence oflarge amounts of non-target DNA was examined. In these experiments amodel target nucleic acid, M13, in either single or double stranded form(single-stranded M13mp18 is available from Life Technologies, Inc anddouble-stranded M 13mp19 is available from New England Biolabs), wascombined with human genomic DNA (Novagen, Madison, Wis.) and thenutilized in invader-directed cleavage reactions. Before the start of thecleavage reaction, the DNAs were heated to 95° C. for 15 minutes tocompletely denature the samples, as is standard practice in assays, suchas polymerase chain reaction or enzymatic DNA sequencing, which involvesolution hybridization of oligonucleotides to double-stranded targetmolecules.

[0492] For each of the reactions shown in lanes 2-5 of FIG. 35, thetarget DNA (25 fmole of the ss DNA or 1 pmole of the ds DNA) wascombined with 50 pmole of the invader oligonucleotide (SEQ ID NO:46);for the reaction shown in lane 1 the target DNA was omitted. Reactions1, 3 and 5 also contained 470 ng of human genomic DNA. These mixtureswere brought to a volume of 10 μl with distilled water, overlaid with adrop of ChillOut™ evaporation barrier (MJ Research), and brought to 95°C. for 15 minutes. After this incubation period, and still at 95° C.,each tube received 10 μl of a mixture comprising 2.25 μl of Cleavase®A/G nuclease extract (prepared as described in Example 2) and 5 pmole ofthe probe oligonucleotide (SEQ ID NO:43), in 20 mM MOPS, pH 7.5 with0.1% each of Tween 20 and NP-40, 4 mM MnCl₂ and 100 mM KCl. Thereactions were brought to 62° C. for 15 minutes and stopped by theaddition of 12 μl of 95% formamide with 20 mM EDTA and 0.05% markerdyes. Samples were heated to 75° C. for 2 minutes immediately beforeelectrophoresis through a 20% acrylamide gel (19:1 cross-linked), with 7M urea, in a buffer of 45 mM Tris-Borate, pH 8.3, 1.4 mM EDTA. Theproducts of the reactions were visualized by the use of an Hitachi FMBIOfluorescence imager. The results are displayed in FIG. 35.

[0493] In FIG. 35, lane 1 contains the products of the reactioncontaining the probe (SEQ ID NO:43), the invader oligonucleotide (SEQ IDNO:46) and human genomic DNA. Examination of lane 1 shows that the probeand invader oligonucleotides are specific for the target sequence, andthat the presence of genomic DNA does not cause any significantbackground cleavage.

[0494] In FIG. 35, lanes 2 and 3 contain reaction products fromreactions containing the single-stranded target DNA (M13mp18), the probe(SEQ ID NO:43) and the invader oligonucleotide (SEQ ID NO:46) in theabsence or presence of human genomic DNA, respectively. Examination oflanes 2 and 3 demonstrate that the invader detection assay may be usedto detect the presence of a specific sequence on a single-strandedtarget molecule in the presence or absence of a large excess ofcompetitor DNA (human genomic DNA).

[0495] In FIG. 35, lanes 4 and 5 contain reaction products fromreactions containing the double-stranded target DNA (M13mp19), the probe(SEQ ID NO:43) and the invader oligonucleotide (SEQ ID NO:46) in theabsence or presence of human genomic DNA, respectively. Examination oflanes 4 and 5 show that double stranded target molecules are eminentlysuitable for invader-directed detection reactions. The success of thisreaction using a short duplexed molecule, M13mp19, as the target in abackground of a large excess of genomic DNA is especially noteworthy asit would be anticipated that the shorter and less complex M13 DNAstrands would be expected to find their complementary strand more easilythan would the strands of the more complex human genomic DNA. If the M13DNA reannealed before the probe and/or invader oligonucleotides couldbind to the target sequences along the M13 DNA, the cleavage reactionwould be prevented. In addition, because the denatured genomic DNA wouldpotentially contain regions complementary to the probe and/or invaderoligonucleotides it was possible that the presence of the genomic DNAwould inhibit the reaction by binding these oligonucleotides therebypreventing their hybridization to the M13 target. The above resultsdemonstrate that these theoretical concerns are not a problem under thereaction conditions employed above.

[0496] In addition to demonstrating that the invader detection assay maybe used to detect sequences present in a double-stranded target, thesedata also show that the presence of a large amount of non-target DNA(470 ng/20 μl reaction) does not lessen the specificity of the cleavage.While this amount of DNA does show some impact on the rate of productaccumulation, probably by binding a portion of the enzyme, the nature ofthe target sequence, whether single- or double-stranded nucleic acid,does not limit the application of this assay.

EXAMPLE 15 Signal Accumulation in the Invader-Directed Cleavage Assay asa Function of Target Concentration

[0497] To investigate whether the invader-directed cleavage assay couldbe used to indicate the amount of target nucleic acid in a sample, thefollowing experiment was performed. Cleavage reactions were assembledwhich contained an invader oligonucleotide (SEQ ID NO:46), a labelledprobe (SEQ ID NO:43) and a target nucleic acid, M13mp19. A series ofreactions, which contained smaller and smaller amounts of the M13 targetDNA, was employed in order to examine whether the cleavage productswould accumulate in a manner that reflected the amount of target DNApresent in the reaction.

[0498] The reactions were conducted as follows. A master mix containingenzyme and buffer was assembled. Each 5 μl of the master mixturecontained 25 ng of Cleavase® BN nuclease in 20 mM MOPS (pH 7.5) with0.1% each of Tween 20 and NP-40, 4 mM MnCl₂ and 100 mM KCl. For each ofthe cleavage reactions shown in lanes 4-13 of FIG. 36, a DNA mixture wasgenerated which contained 5 pmoles of the fluorescein-labelled probeoligonucleotide (SEQ ID NO:43), 50 pmoles of the invader oligonucleotide(SEQ ID NO:46) and 100, 50, 10, 5, 1, 0.5, 0.1, 0.05, 0.01 or 0.005fmoles of single-stranded M13mp19, respectively, for every 5 μl of theDNA mixture. The DNA solutions were covered with a drop of ChillOut®evaporation barrier (MJ Research) and brought to 61° C. The cleavagereactions were started by the addition of 5 μl of the enzyme mixture toeach of tubes (final reaction volume was 10 μl). After 30 minutes at 61°C., the reactions were terminated by the addition of 8 μl of 95%formamide with 20 mM EDTA and 0.05% marker dyes. Samples were heated to90° C. for 1 minutes immediately before electrophoresis through a 20%denaturing acrylamide gel (19:1 cross-linked) with 7 M urea, in a buffercontaining 45 mM Tris-Borate (pH 8.3), 1.4 mM EDTA. To provide reference(i.e., standards), 1.0, 0.1 and 0.01 pmole aliqouts offluorescein-labelled probe oligonucleotide (SEQ ID NO:43) were dilutedwith the above formamide solution to a final volume of 18 μl. Thesereference markers were loaded into lanes 1-3, respectively of the gel.The products of the cleavage reactions (as well as the referencestandards) were visualized following electrophoresis by the use of aHitachi FMBIO fluorescence imager. The results are displayed in FIG. 36.

[0499] In FIG. 36, boxes appear around fluorescein-containing nucleicacid (i.e., the cleaved and uncleaved probe molecules) and the amount offluorescein contained within each box is indicated under the box. Thebackground fluorescence of the gel (see box labelled “background”) wassubtracted by the fluoro-imager to generate each value displayed under abox containing cleaved or uncleaved probe products (the boxes arenumbered 1-14 at top left with a V followed by a number below the box).The lane marked “M” contains fluoresceinated oligonucleotides whichserved as markers.

[0500] The results shown in FIG. 36, demonstrate that the accumulationof cleaved probe molecules in a fixed-length incubation period reflectsthe amount of target DNA present in the reaction. The results alsodemonstrate that the cleaved probe products accumulate in excess of thecopy number of the target. This is clearly demonstrated by comparing theresults shown in lane 3, in which 10 fmole (0.01 pmole) of uncut probeare displayed with the results shown in 5, where the products whichaccumulated in response to the presence of 10 fmole of target DNA aredisplayed. These results show that the reaction can cleave hundreds ofprobe oligonucleotide molecules for each target molecule present,dramatically amplifying the target-specific signal generated in theinvader-directed cleavage reaction.

EXAMPLE 16 Effect of Saliva Extract on the Invader-Directed CleavageAssay

[0501] For a nucleic acid detection method to be useful in a medical(i.e., a diagnostic) setting, it must not be inhibited by materials andcontaminants likely to be found in a typical clinical specimen. To testthe susceptibility of the invader-directed cleavage assay to variousmaterials, including but not limited to nucleic acids, glycoproteins andcarbohydrates, likely to be found in a clinical sample, a sample ofhuman saliva was prepared in a manner consistent with practices in theclinical laboratory and the resulting saliva extract was added to theinvader-directed cleavage assay. The effect of the saliva extract uponthe inhibition of cleavage and upon the specificity of the cleavagereaction was examined.

[0502] One and one-half milliliters of human saliva were collected andextracted once with an equal volume of a mixture containingphenol:chloroform:isoamyl alcohol (25:24:1). The resulting mixture wascentrifuged in a microcentrifuge to separate the aqueous and organicphases. The upper, aqueous phase was transferred to a fresh tube.One-tenth volumes of 3 M NaOAc were added and the contents of the tubewere mixed. Two volumes of 100% ethyl alcohol were added to the mixtureand the sample was mixed and incubated at room temperature for 15minutes to allow a precipitate to form. The sample was centrifuged in amicrocentrifuge at 13,000 rpm for 5 minutes and the supernatant wasremoved and discarded. A milky pellet was easily visible. The pellet wasrinsed once with 70% ethanol, dried under vacuum and dissolved in 200 μlof 10 mM Tris-HCl, pH 8.0, 0.1 mM EDTA (this constitutes the salivaextract). Each μl of the saliva extract was equivalent to 7.5 μl ofsaliva. Analysis of the saliva extract by scanning ultravioletspectrophotometry showed a peak absorbance at about 260 nm and indicatedthe presence of approximately 45 ng of total nucleic acid per μl ofextract.

[0503] The effect of the presence of saliva extract upon the followingenzymes was examined: Cleavase® BN nuclease, Cleavase® A/G nuclease andthree different lots of DNAPTaq: AmpliTaq® (Perkin Elmer; a recombinantform of DNAPTaq), AmpliTaq® LD (Perkin-Elmer; a recombinant DNAPTaqpreparation containing very low levels of DNA) and Taq DNA polymerase(Fischer). For each enzyme tested, an enzyme/probe mixture was madecomprising the chosen amount of enzyme with 5 pmole of the probeoligonucleotide (SEQ ID NO:43) in 10 μl of 20 mM MOPS (pH 7.5)containing 0.1% each of Tween 20 and NP-40, 4 mM MnCl₂, 100 mM KCl and100 μg/ml BSA. The following amounts of enzyme were used: 25 ng ofCleavase® BN prepared as described in Example 9; 2 μl of Cleavase® A/Gnuclease extract prepared as described in Example 2; 2.25 μl (11.25polymerase units) the following DNA polymerases: AmpliTaq® DNApolymerase (Perkin Elmer); AmpliTaq® DNA polymerase LD (low DNA; fromPerkin Elmer); Taq DNA polymerase (Fisher Scientific).

[0504] For each of the reactions shown in FIG. 37, except for that shownin lane 1, the target DNA (50 fmoles of single-stranded M13mp19 DNA) wascombined with 50 pmole of the invader oligonucleotide (SEQ ID NO:46) and5 pmole of the probe oligonucleotide (SEQ ID NO:43); target DNA wasomitted in reaction I (lane 1). Reactions 1, 3, 5, 7, 9 and 11 included1.5 l of saliva extract. These mixtures were brought to a volume of 5 μlwith distilled water, overlaid with a drop of ChillOut® evaporationbarrier (MJ Research) and brought to 95° C. for 10 minutes. The cleavagereactions were then started by the addition of 5 μl of the desiredenzyme/probe mixture; reactions 1, 4 and 5 received Cleavase® A/Gnuclease. Reactions 2 and 3 received Cleavase® BN; reactions 6 and 7received AmpliTaq®; reactions 8 and 9 received AmoliTaq® LD; andreactions 10 and 11 received Taq DNA Polymerase from Fisher Scientific.

[0505] The reactions were incubated at 63° C. for 30 minutes and werestopped by the addition of 6 μl of 95% formamide with 20 mM EDTA and0.05% marker dyes. Samples were heated to 75° C. for 2 minutesimmediately before electrophoresis through a 20% acrylamide gel (19:1cross-linked), with 7 M urea, in a buffer of 45 mM Tris-Borate, pH 8.3,1.4 mM EDTA. The products of the reactions were visualized by the use ofan Hitachi FMBIO fluorescence imager, and the results are displayed inFIG. 37.

[0506] A pairwise comparison of the lanes shown in FIG. 37 without andwith the saliva extract, treated with each of the enzymes, shows thatthe saliva extract has different effects on each of the enzymes. Whilethe Cleavase® BN nuclease and the AmpliTaq® are significantly inhibitedfrom cleaving in these conditions, the Cleavase® A/G nuclease andAmpliTaq® LD display little difference in the yield of cleaved probe.The preparation of Taq DNA polymerase from Fisher Scientific shows anintermediate response, with a partial reduction in the yield of cleavedproduct. From the standpoint of polymerization, the three DNAPTaqvariants should be equivalent; these should be the same protein with thesame amount of synthetic activity. It is possible that the differencesobserved could be due to variations in the amount of nuclease activitypresent in each preparation caused by different handling duringpurification, or by different purification protocols. In any case,quality control assays designed to assess polymerization activity incommercial DNAP preparations would be unlikely to reveal variation inthe amount of nuclease activity present. If preparations of DNAPTaq werescreened for full 5′ nuclease activity (i.e., f the 5′ nuclease activitywas specifically quantitated), it is likely that the preparations woulddisplay sensitivities (to saliva extract) more in line with thatobserved using Cleavase® A/G nuclease, from which DNAPTaq differs by avery few amino acids.

[0507] It is worthy of note that even in the slowed reactions ofCleavase® BN and the DNAPTaq variants there is no noticeable increase innon-specific cleavage of the probe oligonucleotide due to inappropriatehybridization or saliva-borne nucleases.

EXAMPLE 17 Comparison of Additional 5′ Nucleases in the Invader-DirectedCleavage Assay

[0508] A number of eubacterial Type A DNA polymerases (i.e., Pol I typeDNA polymerases) have been shown to function as structure specificendonucleases (Example 1 and Lyamichev et al., supra). In this example,it was demonstrated that the enzymes of this class can also be made tocatalyze the invader-directed cleavage of the present invention, albeitnot as efficiently as the Cleavase® enzymes.

[0509] Cleavase® BN nuclease and Cleavase® A/G nuclease were testedalong side three different thermostable DNA polymerases: Thermusaquaticus DNA polymerase (Promega), Thermus thermophilus and Thermusflavus DNA polymerases (Epicentre). The enzyme mixtures used in thereactions shown in lanes 1-11 of FIG. 38 contained the following, eachin a volume of 5 μl: Lane 1: 20 mM MOPS (pH 7.5) with 0.1% each of Tween20 and NP-40, 4 mM MnCl₂, 100 mM KCl; Lane 2: 25 ng of Cleavase® BNnuclease in the same solution described for lane 1; Lane 3: 2.25 μl ofCleavase® A/G nuclease extract (prepared as described in Example 2), inthe same solution described for lane 1; Lane 4: 2.25 μl of Cleavase®)A/G nuclease extract in 20 mM Tris-Cl, (pH 8.5), 4 mM MgCl₂ and 100 mMKCl; Lane 5: 11.25 polymerase units of Taq DNA polymerase in the samebuffer described for lane 4; Lane 6: 1 11.25 polymerase units of Tth DNApolymerase in the same buffer described for lane 1; Lane 7: 11.25polymerase units of Tth DNA polymerase in a 2× concentration of thebuffer supplied by the manufacturer, supplemented with 4 mM MnCl₂; Lane8: 11.25 polymerase units of Tth DNA polymerase in a 2× concentration ofthe buffer supplied by the manufacturer, supplemented with 4 mM MgCl₂;Lane 9: 2.25 polymerase units of Tfl DNA polymerase in the same bufferdescribed for lane 1; Lane 10: 2.25 polymerase units of Tfl polymerasein a 2× concentration of the buffer supplied by the manufacturer,supplemented with 4 mM MnCl₂; Lane 11: 2.25 polymerase units of Tfl DNApolymerase in a 2× concentration of the buffer supplied by themanufacturer, supplemented with 4 mM MgCl₂.

[0510] Sufficient target DNA, probe and invader for all 11 reactions wascombined into a master mix. This mix contained 550 fmoles ofsingle-stranded M13mp19 target DNA, 550 pmoles of the invaderoligonucleotide (SEQ ID NO:46) and 55 pmoles of the probeoligonucleotide (SEQ ID NO:43), each as depicted in FIG. 32c, in 55 μlof distilled water. Five μl of the DNA mixture was dispensed into eachof 11 labeled tubes and overlaid with a drop of ChillOut® evaporationbarrier (MJ Research). The reactions were brought to 63° C. and cleavagewas started by the addition of 5 μl of the appropriate enzyme mixture.The reaction mixtures were then incubated at 63° C. temperature for 15minutes. The reactions were stopped by the addition of 8 μl of 95%formamide with 20 mM EDTA and 0.05% marker dyes. Samples were heated to90° C. for 1 minute immediately before electrophoresis through a 20%acrylamide gel (19:1 cross-linked), with 7 M urea, in a buffer of 45 mMTris-Borate (pH 8.3), 1.4 mM EDTA. Following electrophoresis, theproducts of the reactions were visualized by the use of an Hitachi FMBIOfluorescence imager, and the results are displayed in FIG. 38.Examination of the results shown in FIG. 38 demonstrates that all of the5′ nucleases tested have the ability to catalyze invader-directedcleavage in at least one of the buffer systems tested. Although notoptimized here, these cleavage agents are suitable for use in themethods of the present invention.

EXAMPLE 18 The Invader-Directed Cleavage Assay can Detect Single BaseDifferences in Target Nucleic Acid Sequences

[0511] The ability of the invader-directed cleavage assay to detectsingle base mismatch mutations was examined. Two target nucleic acidsequences containing Cleavase® enzyme-resistant phosphorothioatebackbones were chemically synthesized and purified by polyacrylamide gelelectrophoresis. Targets comprising phosphorothioate backbones were usedto prevent exonucleolytic nibbling of the target when duplexed with anoligonucleotide. A target oligonucleotide, which provides a targetsequence that is completely complementary to the invader oligonucleotide(SEQ ID NO:46) and the probe oligonucleotide (SEQ ID NO:43), containedthe following sequence: 5′-CCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGC-3′(SEQ ID NO:47). A second target sequence containing a single base changerelative to SEQ ID NO:47 was synthesized:5′-CCTTTCGCTCTCTTCCCTTCCTTTCTCGCC ACGTTCGCCGGC-3 (SEQ ID NO:48; thesingle base change relative to SEQ ID NO:47 is shown using bold andunderlined type). The consequent mismatch occurs within the “Z” regionof the target as represented in FIG. 29.

[0512] To discriminate between two target sequences which differ by thepresence of a single mismatch), invader-directed cleavage reactions wereconducted using two different reaction temperatures (55° C. and 60° C.).Mixtures containing 200 fmoles of either SEQ ID NO:47 or SEQ ID NO:48, 3pmoles of fluorescein-labelled probe oligonucleotide (SEQ ID NO:43), 7.7pmoles of invader oligonucleotide (SEQ ID NO:46) and 2 μl of Cleavase®A/G nuclease extract (prepared as described in Example 2) in 9 μl of 10mM MOPS (pH 7.4) with 50 mM KCl were assembled, 0 covered with a drop ofChillOut® evaporation barrier (MJ Research) and brought to theappropriate reaction temperature. The cleavage reactions were initiatedby the addition of 1 μl of 20 mM MgCl₂. After 30 minutes at either 55°C. or 60° C., 10 μl of 95% formamide with 20 mM EDTA and 0.05% markerdyes was added to stop the reactions. The reaction mixtures where thenheated to 90° C. for one minute prior to loading 4 μl onto 20%denaturing polyacrylamide gels. The resolved reaction products werevisualized using a Hitachi FMBIO fluorescence imager. The resultingimage is shown in FIG. 39.

[0513] In FIG. 39, lanes 1 and 2 show the products from reactionsconducted at 55° C.; lanes 3 and 4 show the products from reactionsconducted at 60° C. Lanes 1 and 3 contained products from reactionscontaining SEQ ID NO:47 (perfect match to probe) as the target. Lanes 2and 4 contained products from reactions containing SEQ ID NO:48 (singlebase mis-match with probe) as the target. The target that does not havea perfect hybridization match (i.e., complete complementarity) with theprobe will not bind as strongly, i.e., the T_(m) of that duplex will belower than the T_(m) of the same region if perfectly matched. Theresults presented here show that reaction conditions can be varied toeither accommodate the mis-match (e.g., by lowering the temperature ofthe reaction) or to exclude the binding of the mismatched sequence(e.g., by raising the reaction temperature).

[0514] The results shown in FIG. 39 demonstrate that the specificcleavage event which occurs in invader-directed cleavage reactions canbe eliminated by the presence of a single base mis-match between theprobe oligonucleotide and the target sequence. Thus, reaction conditionscan be chosen so as to exclude the hybridization of mis-matchedinvader-directed cleavage probes thereby diminishing or even eliminatingthe cleavage of the probe. In an extension of this assay system,multiple cleavage probes, each possessing a separate reporter molecule(i.e., a unique label), could also be used in a single cleavagereaction, to simultaneously probe for two or more variants in the sametarget region. The products of such a reaction would allow not only thedetection of mutations which exist within a target molecule, but wouldalso allow a determination of the relative concentrations of eachsequence (i.e., mutant and wild type or multiple different mutants)present within samples containing a mixture of target sequences. Whenprovided in equal amounts, but in a vast excess (e.g., at least a100-fold molar excess; typically at least 1 pmole of each probeoligonucleotide would be used when the target sequence was present atabout 10 fmoles or less) over the target and used in optimizedconditions. As discussed above, any differences in the relative amountsof the target variants will not affect the kinetics of hybridization, sothe amounts of cleavage of each probe will reflect the relative amountsof each variant present in the reaction.

[0515] The results shown in the example clearly demonstrate that theinvader-directed cleavage reaction can be used to detect single basedifference between target nucleic acids.

EXAMPLE 19 The Invader-Directed Cleavage Reaction is Insensitive toLarge Changes in Reaction Conditions

[0516] The results shown above demonstrated that the invader-directedcleavage reaction can be used for the detection of target nucleic acidsequences and that this assay can be used to detect single basedifference between target nucleic acids. These results demonstrated that5′ nucleases (e.g., Cleavase®BN, Cleavase® A/G, DNAPTaq, DNAPTth,DNAPTfl) could be used in conjunction with a pair of overlappingoligonucleotides as an efficient way to recognize nucleic acid targets.In the experiments below it is demonstrated that invasive cleavagereaction is relatively insensitive to large changes in conditionsthereby making the method suitable for practice in clinicallaboratories.

[0517] The effects of varying the conditions of the cleavage reactionwere examined for their effect(s) on the specificity of the invasivecleavage and the on the amount of signal accumulated in the course ofthe reaction. To compare variations in the cleavage reaction a“standard” invader cleavage reaction was first defined. In eachinstance, unless specifically stated to be otherwise, the indicatedparameter of the reaction was varied, while the invariant aspects of aparticular test were those of this standard reaction. The results ofthese tests are shown in FIGS. 42-51.

[0518] a) The Standard Invader-Directed Cleavage Reaction

[0519] The standard reaction was defined as comprising 1 fmole ofM13mp18 single-stranded target DNA (New England Biolabs), 5 pmoles ofthe labeled probe oligonucleotide (SEQ ID NO:49), 10 pmole of theupstream invader oligonucleotide (SEQ ID NO:50) and 2 units of Cleavase®A/G in 10 μl of 10 mM MOPS, pH 7.5 with 100 mM KCl, 4 mM MnCl₂, and0.05% each Tween-20 and Nonidet-P40. For each reaction, the buffers,salts and enzyme were combined in a volume of 5 μl; the DNAs (target andtwo oligonucleotides) were combined in 5 μl of dH₂O and overlaid with adrop of ChillOut® evaporation barrier (MJ Research). When multiplereactions were performed with the same reaction constituents, theseformulations were expanded proportionally.

[0520] Unless otherwise stated, the sample tubes with the DNA mixtureswere warmed to 61° C., and the reactions were started by the addition of5 μl of the enzyme mixture. After 20 minutes at this temperature, thereactions were stopped by the addition of 8 μl of 95% formamide with 20mM EDTA and 0.05% marker dyes. Samples were heated to 75° C. for 2minutes immediately before electrophoresis through a 20% acrylamide gel(19:1 cross-linked), with 7 M urea, in a buffer of 45 mM Tris-Borate, pH8.3, 1.4 mM EDTA. The products of the reactions were visualized by theuse of an Hitachi FMBIO fluorescence imager. In each case, the uncutprobe material was visible as an intense black band or blob, usually inthe top half of the panel, while the desired products of invaderspecific cleavage were visible as one or two narrower black bands,usually in the bottom half of the panel. Under some reaction conditions,particulary those with elevated salt concentrations, a secondarycleavage product is also visible (thus generating a doublet). Ladders oflighter grey bands generally indicate either exonuclease nibbling of theprobe oligonucleotide or heat-induced, non-specific breakage of theprobe.

[0521]FIG. 41 depicts the annealing of the probe and invaderoligonucleotides to regions along the M13mp18 target molecule (thebottom strand). In FIG. 41 only a 52 nucleotide portion of the M13mp18molecule is shown; this 52 nucleotide sequence is listed in SEQ ID NO:42(this sequence is identical in both M13mp18 and M13mp19). The probeoligonucleotide (top strand) contains a Cy3 amidite label at the 5′ end;the sequence of the probe is 5′-AGAAAGGAAGGGAAGAAAGCGAAA GGT-3′ (SEQ IDNO:49. The bold type indicates the presence of a modified base(2′-O—CH₃). Cy3 amidite (Pharmacia) is a indodicarbocyanine dye amiditewhich can be incorporated at any position during the synthesis ofoligonucleotides; Cy3 fluoresces in the yellow region (excitation andemission maximum of 554 and 568 nm, respectively). The invaderoligonucleotide (middle strand) has the following sequence:5′-GCCGGCGAACGTGGCGAGAAAGGA-3′ (SEQ ID NO:50).

[0522] b) KCl Titration

[0523]FIG. 42 shows the results of varying the KCl concentration incombination with the use of 2 mM MnCl₂, in an otherwise standardreaction. The reactions were performed in duplicate for confirmation ofobservations; the reactions shown in lanes 1 and 2 contained no addedKCl, lanes 3 and 4 contained KCl at 5 mM, lanes 5 and 6 contained 25 mMKCl, lanes 7 and 8 contained 50 mM KCl, lanes 9 and 10 contained 100 mMKCl and lanes 11 and 12 contained 200 mM KCl. These results show thatthe inclusion of KCl allows the generation of a specific cleavageproduct. While the strongest signal is observed at the 100 mM KClconcentration, the specificity of signal in the other reactions with KClat or above 25 mM indicates that concentrations in the full range (i.e.,25-200 mM) may be chosen if it is so desirable for any particularreaction conditions.

[0524] As shown in FIG. 42, the invader-directed cleavage reactionrequires the presence of salt (e.g., KCl) for effective cleavage tooccur. In other reactions, it has been found that KCl can inhibit theactivity of certain Cleavase® enzymes when present at concentrationsabove about 25 mM (For example, in cleavage reactions using the S-60oligonucleotide shown in FIG. 30, in the absence of primer, theCleavase® BN enzyme loses approximately 50% of its activity in 50 mMKCl). Therefore, the use of alternative salts in the invader-directedcleavage reaction was examined. In these experiments, the potassium ionwas replaced with either Na⁺ or Li⁺ or the chloride ion was replacedwith glutamic acid. The replacement of KCl with alternative salts isdescribed below in sections c-e.

[0525] c) NaCl Titration

[0526]FIG. 43 shows the results of using various concentrations of NaClin place of KCl (lanes 3-10) in combination with the use 2 mM MnCl₂, inan otherwise standard reaction, in comparison to the effects seen with100 mM KCl (lanes 1 and 2). The reactions analyzed in lanes 3 and 4contained NaCl at 75 mM, lanes 5 and 6 contained 100 mM, lanes 7 and 8contained 150 mM and lanes 9 and 10 contained 200 mM. These results showthat NaCl can be used as a replacement for KCl in the invader-directedcleavage reaction (i.e., the presence of NaCl, like KCl, enhancesproduct accumulation).

[0527] d) LiCl Titration

[0528]FIG. 44 shows the results of using various concentrations of LiClin place of KCl (lanes 3-14) in otherwise standard reactions, comparedto the effects seen with 100 mM KCl (lanes 1 and 2). The reactionsanalyzed in lanes 3 and 4 contained LiCl at 25 mM, lanes 5 and 6contained 50 mM, lanes 7 and 8 contained 75 mM, lanes 9 and 10 contained100 mM, lanes 11 and 12 contained 150 mM and lanes 13 and 14 contained200 mM. These results demonstrate that LiCl can be used as a suitablereplacement for KCl in the invader-directed cleavage reaction (i.e., thepresence of LiCl, like KCl, enhances product accumulation).

[0529] e) KGlu Titration

[0530]FIG. 45 shows the results of using a glutamate salt of potassium(KGlu) in place of the more commonly used chloride salt (KCl) inreactions performed over a range of temperatures. KGlu has been shown tobe a highly effective salt source for some enzymatic reactions, showinga broader range of concentrations which permit maximum enzymaticactivity [Leirmo et al. (1987) Biochem. 26:2095]. The ability of KGlu tofacilitate the annealing of the probe and invader oligonucleotides tothe target nucleic acid was compared to that of LiCl. In theseexperiments, the reactions were run for 15 minutes, rather than thestandard 20 minutes. The reaction analyzed in lane 1 contained 150 mMLiCl and was run at 65° C.; the reactions analyzed in lanes 2-4contained 200 mM, 300 mM and 400 mM KGlu, respectively and were run at65° C. The reactions analyzed in lanes 5-8 repeated the array of saltconcentrations used in lanes 1-4, but were performed at 67° C.; lanes9-12 show the same array run at 69° C. and lanes 13-16 show the samearray run at 71° C. The results shown in FIG. 45 demonstrate that KGluwas very effective as a salt in the invasive cleavage reactions. Inaddition, these data show that the range of allowable KGluconcentrations was much greater than that of LiCl, with full activityapparent even at 400 mM KGlu.

[0531] f) MnCl₂ and MgCl₂ Titration and Ability to Replace MnCl₂ withMgCl₂

[0532] In some instances it may be desirable to perform the invasivecleavage reaction in the presence of Mg²⁺, either in addition to, or inplace of Mn²⁺ as the necessary divalent cation required for activity ofthe enzyme employed. For example, some common methods of preparing DNAfrom bacterial cultures or tissues use MgCl₂ in solutions which are usedto facilitate the collection of DNA by precipitation. In addition,elevated concentrations (i.e., greater than 5 mM) of divalent cation canbe used to facilitate hybridization of nucleic acids, in the same waythat the monovalent salts were used above, thereby enhancing theinvasive cleavage reaction. In this experiment, the tolerance of theinvasive cleavage reaction was examined for 1) the substitution of MgCl₂for MnCl₂ and for the ability to produce specific product in thepresence of increasing concentrations of MgCl₂ and MnCl₂.

[0533]FIG. 46 shows the results of either varying the concentration ofMnCl₂ from 2 mM to 8 mM, replacing the MnCl₂ with MgCl₂ at 2 to 4 mM, orof using these components in combination in an otherwise standardreaction. The reactions analyzed in lanes 1 and 2 contained 2 mM eachMnCl₂ and MgCl₂, lanes 3 and 4 contained 2 mM MnCl₂ only, lanes 5 and 6contained 3 mM MnCl₂, lanes 7 and 8 contained 4 mM MnCl₂, lanes 9 and 10contained 8 mM MnCl₂. The reactions analyzed in lanes 11 and 12contained 2 mM MgCl₂ and lanes 13 and 14 contained 4 mM MgCl₂. Theseresults show that both MnCl₂ and MgCl₂ can be used as the necessarydivalent cation to enable the cleavage activity of the Cleavase® A/Genzyme in these reactions and that the invasive cleavage reaction cantolerate a broad range of concentrations of these components.

[0534] In addition to examining the effects of the salt environment onthe rate of product accumulation in the invasive cleavage reaction, theuse of reaction constituents shown to be effective in enhancing nucleicacid hybridization in either standard hybridization assays (e.g., blothybridization) or in ligation reactions was examined. These componentsmay act as volume excluders, increasing the effective concentration ofthe nucleic acids of interest and thereby enhancing hybridization, orthey may act as charge-shielding agents to minimize repulsion betweenthe highly charged backbones of the nucleic acids strands. The resultsof these experiments are described in sections g and h below.

[0535] g) Effect of CTAB Addition

[0536] The polycationic detergent cetyltrietheylammonium bromide (CTAB)has been shown to dramatically enhance hybridization of nucleic acids[Pontius and Berg (1991) Proc. Natl. Acad. Sci. USA 88:8237]. The datashown in FIG. 47 depicts the results of adding the detergent CTAB toinvasive cleavage reactions in which 150 mM LiCl was used in place ofthe KCl in otherwise standard reactions. Lane 1 shows unreacted (i.e.,uncut) probe, and the reaction shown in lane 1 is the LiCl-modifiedstandard reaction without CTAB. The reactions analyzed in lanes 3 and 4contained 100 μM CTAB, lanes 5 and 6 contained 200 μM CTAB, lanes 7 and8 contained 400 μM CTAB, lanes 9 and 10 contained 600 μM CTAB, lanes 11and 12 contained 800 μM CTAB and lanes 13 and 14 contained 1 mM CTAB.These results showed that the lower amounts of CTAB may have a verymoderate enhancing effect under these reaction conditions, and thepresence of CTAB in excess of about 500 μM was inhibitory to theaccumulation of specific cleavage product.

[0537] h) Effect of PEG Addition

[0538]FIG. 48 shows the effect of adding polyethylene glycol (PEG) atvarious percentage (w/v) concentrations to otherwise standard reactions.The effects of increasing the reaction temperature of the PEG-containingreactions was also examined. The reactions assayed in lanes 1 and 2 werethe standard conditions without PEG, lanes 3 and 4 contained 4% PEG,lanes 5 and 6 contained 8% PEG and lanes 7 and 8 contained 12% PEG. Eachof the aforementioned reactions was performed at 61° C. The reactionsanalyzed in lanes 9, 10, 11 and 12 were performed at 65° C., andcontained 0%, 4%, 8% and 12% PEG, respectively. These results show thatat all percentages tested, and at both temperatures tested, theinclusion of PEG substantially eliminated the production of specificcleavage product.

[0539] In addition to the data presented above (i.e., effect of CTAB andPEG addition), the presence of 1× Denhardts in the reaction mixture wasfound to have no adverse effect upon the cleavage reaction [SOXDenhardt's contains per 500 ml: 5 g Ficoll, 5 g polyvinylpyrrolidone, 5g BSA]. In addition, the presence of each component of Denhardt's wasexamined individually (i.e., Ficoll alone, polyvinylpyrrolidone alone,BSA alone) for the effect upon the invader-directed cleavage reaction;no adverse effect was observed.

[0540] i) Effect of the Addition of Stabilizing Agents

[0541] Another approach to enhancing the output of the invasive cleavagereaction is to enhance the activity of the enzyme employed, either byincreasing its stability in the reaction environment or by increasingits turnover rate. Without regard to the precise mechanism by whichvarious agents operate in the invasive cleavage reaction, a number ofagents commonly used to stabilize enzymes during prolonged storage weretested for the ability to enhance the accumulation of specific cleavageproduct in the invasive cleavage reaction.

[0542]FIG. 49 shows the effects of adding glycerol at 15% and of addingthe detergents Tween-20 and Nonidet-P40 at 1.5%, alone or incombination, in otherwise standard reactions. The reaction analyzed inlane 1 was a standard reaction. The reaction analyzed in lane 2contained 1.5% NP-40, lane 3 contained 1.5% Tween 20, lane 4 contained15% glycerol. The reaction analyzed in lane 5 contained both Tween-20and NP-40 added at the above concentrations, lane 6 contained bothglycerol and NP-40, lane 7 contained both glycerol and Tween-20, andlane 8 contained all three agents. The results shown in FIG. 49demonstrate that under these conditions these adducts had little or noeffect on the accumulation of specific cleavage product.

[0543]FIG. 50 shows the effects of adding gelatin to reactions in whichthe salt identity and concentration were varied from the standardreaction. In addition, all of these reactions were performed at 65° C.,instead of 61° C. The reactions assayed in lanes 1-4 lacked added KCl,and included 0.02%, 0.05%, 0.1% or 0.2% gelatin, respectively. Lanes 5,6, 7 and 8 contained the same titration of gelatin, respectively, andincluded 100 mM KCl. Lanes 9, 10, 11 and 12, also had the same titrationof gelatin, and additionally included 150 mM LiCl in place of KCl. Lanes13 and 14 show reactions that did not include gelatin, but whichcontained either 100 mM KCl or 150 mM LiCl, respectively. The resultsshown in FIG. 50 demonstrated that in the absence of salt the gelatinhad a moderately enhancing effect on the accumulation of specificcleavage product, but when either salt (KCl or LiCl) was added toreactions performed under these conditions, increasing amounts ofgelatin reduced the product accumulation.

[0544] j) Effect of Adding Large Amounts of Non-Target Nucleic Acid

[0545] In detecting specific nucleic acid sequences within samples, itis important to determine if the presence of additional genetic material(i.e., non-target nucleic acids) will have a negative effect on thespecificity of the assay. In this experiment, the effect of includinglarge amounts of non-target nucleic acid, either DNA or RNA, on thespecificity of the invasive cleavage reaction was examined. The data wasexamined for either an alteration in the expected site of cleavage, orfor an increase in the nonspecific degradation of the probeoligonucleotide.

[0546]FIG. 51 shows the effects of adding non-target nucleic acid (e.g.,genomic DNA or tRNA) to an invasive cleavage reaction performed at 65°C., with 150 mM LiCl in place of the KCl in the standard reaction. Thereactions assayed in lanes 1 and 2 contained 235 and 470 ng of genomicDNA, respectively. The reactions analyzed in lanes 3, 4, 5 and 6contained 100 ng, 200 ng, 500 ng and 1 μg of tRNA, respectively. Lane 7represents a control reaction which contained no added nucleic acidbeyond the amounts used in the standard reaction. The results shown inFIG. 51 demonstrate that the inclusion of non-target nucleic acid inlarge amounts could visibly slow the accumulation of specific cleavageproduct (while not limiting the invention to any particular mechanism,it is thought that the additional nucleic acid competes for binding ofthe enzyme with the specific reaction components). In additionalexperiments it was found that the effect of adding large amounts ofnon-target nucleic acid can be compensated for by increasing the enzymein the reaction. The data shown in FIG. 51 also demonstrate that a keyfeature of the invasive cleavage reaction, the specificity of thedetection, was not compromised by the presence of large amounts ofnon-target nucleic acid.

[0547] In addition to the data presented above, invasive cleavagereactions were run with succinate buffer at pH 5.9 in place of the MOPSbuffer used in the “standard” reaction; no adverse effects wereobserved.

[0548] The data shown in FIGS. 42-51 and described above demonstratethat the invasive cleavage reaction can be performed using a widevariety of reaction conditions and is therefore suitable for practice inclinical laboratories.

EXAMPLE 20 Detection of RNA Targets by Invader-Directed Cleavage

[0549] In addition to the clinical need to detect specific DNA sequencesfor infectious and genetic diseases, there is a need for technologiesthat can quantitatively detect target nucleic acids that are composed ofRNA. For example, a number of viral agents, such as hepatitis C virus(HCV) and human immunodeficiency virus (HIV) have RNA genomic material,the quantitative detection of which can be used as a measure of viralload in a patient sample. Such information can be of critical diagnosticor prognostic value.

[0550] Hepatitis C virus (HCV) infection is the predominant cause ofpost-transfusion non-A, non-B (NANB) hepatitis around the world. Inaddition, HCV is the major etiologic agent of hepatocellular carcinoma(HCC) and chronic liver disease world wide. The genome of HCV is a small(9.4 kb) RNA molecule. In studies of transmission of HCV by bloodtransfusion it has been found the presence of HCV antibody, as measuredin standard immunological tests, does not always correlate with theinfectivity of the sample, while the presence of HCV RNA in a bloodsample strongly correlates with infectivity. Conversely, serologicaltests may remain negative in immunosuppressed infected individuals,while HCV RNA may be easily detected [J. A. Cuthbert (1994) Clin.Microbiol. Rev. 7:505].

[0551] The need for and the value of developing a probe-based assay forthe detection the HCV RNA is clear. The polymerase chain reaction hasbeen used to detect HCV in clinical samples, but the problems associatedwith carry-over contamination of samples has been a concern. Directdetection of the viral RNA without the need to perform either reversetranscription or amplification would allow the elimination of several ofthe points at which existing assays may fail.

[0552] The genome of the positive-stranded RNA hepatitis C viruscomprises several regions including 5′ and 3′ noncoding regions (i.e.,5′ and 3′ untranslated regions) and a polyprotein coding region whichencodes the core protein (C), two envelope glycoproteins (E1 and E2/NS1)and six nonstructural glycoproteins (NS2-NS5b). Molecular biologicalanalysis of the HCV genome has showed that some regions of the genomeare very highly conserved between isolates, while other regions arefairly rapidly changeable. The 5′ noncoding region (NCR) is the mosthighly conserved region in the HCV. These analyses have allowed theseviruses to be divided into six basic genotype groups, and then furtherclassified into over a dozen sub-types [the nomenclature and division ofHCV genotypes is evolving; see Altamirano et al., J. Infect. Dis.171:1034 (1995) for a recent classification scheme].

[0553] In order to develop a rapid and accurate method of detecting HCVpresent in infected individuals, the ability of the invader-directedcleavage reaction to detect HCV RNA was examined. Plasmids containingDNA derived from the conserved 5′-untranslated region of six differentHCV RNA isolates were used to generate templates for in vitrotranscription. The HCV sequences contained within these six plasmidsrepresent genotypes 1 (four sub-types represented; 1a, 1b, 1c, and Δ1c),2, and 3. The nomenclature of the HCV genotypes used herein is that ofSimmonds et al. [as described in Altamirano et at., supra]. The Δ1csubtype was used in the model detection reaction described below.

[0554] a) Generation of Plasmids Containing HCV Sequences

[0555] Six DNA fragments derived from HCV were generated by RT-PCR usingRNA extracted from serum samples of blood donors; these PCR fragmentswere a gift of Dr. M. Altamirano (University of British Columbia.Vancouver). These PCR fragments represent HCV sequences derived from HCVgenotypes 1a, 1b, 1c, Δ1c, 2c and 3a.

[0556] The RNA extraction, reverse transcription and PCR were performedusing standard techniques (Altamirano et al., supra). Briefly, RNA wasextracted from 100 μl of serum using guanidine isothiocyanate, sodiumlauryl sarkosate and phenol-chloroform [Inchauspe et al., Hepatology14:595 (1991)]. Reverse transcription was performed according to themanufacturer's instructions using a GeneAmp rTh reverse transcriptaseRNA PCR kit (Perkin-Elmer) in the presence of an external antisenseprimer, HCV342. The sequence of the HCV342 primer is 5′-GGTTTTTCTTTGAGGTTTAG-3′ (SEQ ID NO:51). Following termination of the RT reaction, thesense primer HCV7 [5′-GCGACACTCCACCATAGAT-3′ (SEQ ID NO:52)] andmagnesium were added and a first PCR was performed. Aliquots of thefirst PCR products were used in a second (nested) PCR in the presence ofprimers HCV46 [5′-CTGTCTTCACGCAGAAAGC-3′ (SEQ ID NO:53)] and HCV308[5′-GCACGGT CTACGAGACCTC-3′ (SEQ ID NO:54)]. The PCRs produced a 281 bpproduct which corresponds to a conserved 5′ noncoding region (NCR)region of HCV between positions −284 and −4 of the HCV genome(Altramirano et al., supra).

[0557] The six 281 bp PCR fragments were used directly for cloning orthey were subjected to an additional amplification step using a 50 μlPCR comprising approximately 100 fmoles of DNA, the HCV46 and HCV308primers at 0.1 μM, 100 μM of all four dNTPs and 2.5 units of Taq DNApolymerase in a buffer containing 10 mM Tris-HCl, pH 8.3, 50 mM KCl, 1.5mM MgCl₂ and 0.1% Tween 20. The PCRs were cycled 25 times at 96° C. for45 sec., 55° C. for 45 sec. and 72° C. for 1 min. Two microliters ofeither the original DNA samples or the reamplified PCR products wereused for cloning in the linear pT7Blue T-vector (Novagen, Madison, Wis.)according to manufacturer's protocol. After the PCR products wereligated to the pT7Blue T-vector, the ligation reaction mixture was usedto transform competent JM109 cells (Promega). Clones containing thepT7Blue T-vector with an insert were selected by the presence ofcolonies having a white color on LB plates containing 40 μg/ml X-Gal, 40μg/ml IPTG and 50 μg/ml ampicillin. Four colonies for each PCR samplewere picked and grown overnight in 2 ml LB media containing 50 μg/mlcarbenicillin. Plasmid DNA was isolated using the following alkalineminiprep protocol. Cells from 1.5 ml of the overnight culture werecollected by centrifugation for 2 min. in a microcentrifuge (14K rpm),the supernatant was discarded and the cell pellet was resuspended in 50μl TE buffer with 10 μg/ml RNAse A (Pharmacia). One hundred microlitersof a solution containing 0.2 N NaOH, 1% SDS was added and the cells werelysed for 2 min. The lysate was gently mixed with 100 μl of 1.32 Mpotassium acetate, pH 4.8, and the mixture was centrifuged for 4 min. ina microcentrifuge (14K rpm); the pellet comprising cell debris wasdiscarded. Plasmid DNA was precipitated from the supernatant with 200 μlethanol and pelleted by centrifugation a microcentrifuge (14K rpm). TheDNA pellet was air dried for 15 min. and was then redissolved in 50 μlTE buffer (10 mM Tris-HCl, pH 7.8, 1 mM EDTA).

[0558] b) Reamplification of HCV Clones to Add the Phage T7 Promoter forSubsequent in Vitro Transcription

[0559] To ensure that the RNA product of transcription had a discrete 3′end it was necessary to create linear transcription templates whichstopped at the end of the HCV sequence. These fragments wereconveniently produced using the PCR to reamplify the segment of theplasmid containing the phage promoter sequence and the HCV insert. Forthese studies, the clone of HCV type Δ1c was reamplified using a primerthat hybridizes to the T7 promoter sequence: 5′-TAATACGACTCACTATAGGG-3′(SEQ ID NO:55; “the T7 promoter primer”) (Novagen) in combination withthe 3′ terminal HCV-specific primer HCV308 (SEQ ID NO:54). For thesereactions, 1 μl of plasmid DNA (approximately 10 to 100 ng) wasreamplified in a 200 μl PCR using the T7 and HCV308 primers as describedabove with the exception that 30 cycles of amplification were employed.The resulting amplicon was 354 bp in length. After amplification the PCRmixture was transferred to a fresh 1.5 ml microcentrifuge tube, themixture was brought to a final concentration of 2 M NH₄₀Ac, and theproducts were precipitated by the addition of one volume of 100%isopropanol. Following a 10 min. incubation at room temperature, theprecipitates were collected by centrifugation, washed once with 80%ethanol and dried under vacuum. The collected material was dissolved in100 μl nuclease-free distilled water (Promega).

[0560] Segments of RNA were produced from this amplicon by in vitrotranscription using the RiboMAX™ Large Scale RNA Production System(Promega) in accordance with the manufacturer's instructions, using 5.3μg of the amplicon described above in a 100 μl reaction. Thetranscription reaction was incubated for 3.75 hours, after which the DNAtemplate was destroyed by the addition of 5-6 μl of RQ1 RNAse-free DNAse(1 unit/μl) according to the RiboMAX™ kit instructions. The reaction wasextracted twice with phenol/chloroform/isoamyl alcohol (50:48:2) and theaqueous phase was transferred to a fresh microcentrifuge tube. The RNAwas then collected by the addition of 10 μl of 3M NH₄₀Ac, pH 5.2 and 110μl of 100% isopropanol. Following a 5 min. incubation at 4° C., theprecipitate was collected by centrifugation, washed once with 80%ethanol and dried under vacuum. The sequence of the resulting RNAtranscript (HCV1.1 transcript) is listed in SEQ ID NO:56.

[0561] c) Detection of the HCV1.1 Transcript in the Invader-DirectedCleavage Assay

[0562] Detection of the HCV1.1 transcript was tested in theinvader-directed cleavage assay using an HCV-specific probeoligonucleotide [5′-CCGGTCGTCCTGGCAAT XCC-3′ (SEQ ID NO:57); X indicatesthe presence of a fluorescein dye on an abasic linker) and anHCV-specific invader oligonucleotide [5′-GTTTATCCAAGAAAGGAC CCGGTCC-3′(SEQ ID NO:58)] that causes a 6-nucleotide invasive cleavage of theprobe.

[0563] Each 10 μl of reaction mixture comprised 5 pmole of the probeoligonucleotide (SEQ ID NO:57) and 10 pmole of the invaderoligonucleotide (SEQ ID NO:58) in a buffer of 10 mM MOPS, pH 7.5 with 50mM KCl, 4 mM MnCl₂, 0.05% each Tween-20 and Nonidet-P40 and 7.8 unitsRNasin® ribonuclease inhibitor (Promega). The cleavage agents employedwere Cleavase® A/G (used at 5.3 ng/10 μl reaction) or DNAPTth (used at 5polymerase units/10 μl reaction). The amount of RNA target was varied asindicated below. When RNAse treatment is indicated, the target RNAs werepre-treated with 10 μg of RNase A (Sigma) at 37° C. for 30 min. todemonstrate that the detection was specific for the RNA in the reactionand not due to the presence of any residual DNA template from thetranscription reaction. RNase-treated aliquots of the HCV RNA were useddirectly without intervening purification.

[0564] For each reaction, the target RNAs were suspended in the reactionsolutions as described above, but lacking the cleavage agent and theMnCl₂ for a final volume of 10 μl, with the invader and probe at theconcentrations listed above. The reactions were warmed to 46° C. and thereactions were started by the addition of a mixture of the appropriateenzyme with MnCl₂. After incubation for 30 min. at 46° C., the reactionswere stopped by the addition of 8 μl of 95% formamide, 10 mM EDTA and0.02% methyl violet (methyl violet loading buffer). Samples were thenresolved by electrophoresis through a 15% denaturing polyacrylamide gel(19:1 cross-linked), containing 7 M urea, in a buffer of 45 mMTris-Borate, pH 8.3, 1.4 mM EDTA. Following electrophoresis, the labeledreaction products were visualized using the FMBIO-100 Image Analyzer(Hitachi), with the resulting imager scan shown in FIG. 52.

[0565] In FIG. 52, the samples analyzed in lanes 1-4 contained 1 pmoleof the RNA target, the reactions shown in lanes 5-8 contained 100 fmolesof the RNA target and the reactions shown in lanes 9-12 contained 10fmoles of the RNA target. All odd-numbered lanes depict reactionsperformed using Cleavase® A/G enzyme and all even-numbered lanes depictreactions performed using DNAPTth. The reactions analyzed in lanes 1, 2,5, 6, 9 and 10 contained RNA that had been pre-digested with RNase A.These data demonstrate that the invasive cleavage reaction efficientlydetects RNA targets and further, the absence of any specific cleavagesignal in the RNase-treated samples confirms that the specific cleavageproduct seen in the other lanes is dependent upon the presence of inputRNA.

EXAMPLE 21 The Fate of the Target RNA in the Invader-Directed CleavageReaction

[0566] In this example, the fate of the RNA target in theinvader-directed cleavage reaction was examined. As shown above inExample ID, when RNAs are hybridized to DNA oligonucleotides, the 5′nucleases associated with DNA polymerases can be used to cleave theRNAs; such cleavage can be suppressed when the 5′ arm is long or when itis highly structured [Lyamichev et al. (1993) Science 260:778 and U.S.Pat. No. 5,422,253, the disclosure of which is herein incorporated byreference]. In this experiment, the extent to which the RNA target wouldbe cleaved by the cleavage agents when hybridized to the detectionoligonucleotides (i.e., the probe and invader oligonucleotides) wasexamined using reactions similar to those described in Example 20,performed using fluorescein-labeled RNA as a target.

[0567] Transcription reactions were performed as described in Example 20with the exception that 2% of the UTP in the reaction was replaced withfluorescein-12-UTP (Boehringer Mannheim) and 5.3 μg of the amplicon wasused in a 100 μl reaction. The transcription reaction was incubated for2.5 hours, after which the DNA template was destroyed by the addition of5-6 μl of RQ1 RNAse-free DNAse (1 unit/l) according to the RiiboMAX™ kitinstructions. The organic extraction was omitted and the RNA wascollected by the addition of 10 μl of 3M NaOAc, pH 5.2 and 110 μl of100% isopropanol. Following a 5 min. incubation at 4° C., theprecipitate was collected by centrifugation, washed once with 80%ethanol and dried under vacuum. The resulting RNA was dissolved in 100μl of nuclease-free water. 50% of the sample was purified byelectrophoresis through a 8% denaturing polyacrylamide gel (19:1cross-linked), containing 7 M urea, in a buffer of 45 mM Tris-Borate, pH8.3, 1.4 mM EDTA. The gel slice containing the full-length material wasexcised and the RNA was eluted by soaking the slice overnight at 4° C.in 200 μl of 10 mM Tris-Cl, pH 8.0, 0.1 mM EDTA and 0.3 M NaOAc. The RNAwas then precipitated by the addition of 2.5 volumes of 100% ethanol.After incubation at −20° C. for 30 min., the precipitates were recoveredby centrifugation, washed once with 80% ethanol and dried under vacuum.The RNA was dissolved in 25 μl of nuclease-free water and thenquantitated by UV absorbance at 260 nm.

[0568] Samples of the purified RNA target were incubated for 5 or 30min. in reactions that duplicated the Cleavase® A/G and DNAPTth invaderreactions described in Example 20 with the exception that the reactionslacked probe and invader oligonucleotides. Subsequent analysis of theproducts showed that the RNA was very stable, with a very slightbackground of non-specific degradation, appearing as a gray backgroundin the gel lane. The background was not dependent on the presence ofenzyme in the reaction.

[0569] Invader detection reactions using the purified RNA target wereperformed using the probe/invader pair described in Example 20 (SEQ IDNOS:57 and 58). Each reaction included 500 fmole of the target RNA, 5pmoles of the fluorescein-labeled probe and 10 pmoles of the invaderoligonucleotide in a buffer of 10 mM MOPS, pH 7.5 with 150 mM LiCl, 4 mMMnCl₂, 0.05% each Tween-20 and Nonidet-P40 and 39 units RNAsin®(Promega). These components were combined and warmed to 50° C. and thereactions were started by the addition of either 53 ng of Cleavase® A/Gor 5 polymerase units of DNAPTth. The final reaction volume was 10 μl.After 5 min at 50° C., 5 μl aliquots of each reaction were removed totubes containing 4 μl of 95% formamide, 10 mM EDTA and 0.02% methylviolet. The remaining aliquot received a drop of ChillOut® evaporationbarrier and was incubated for an additional 25 min. These reactions werethen stopped by the addition of 4 μl of the above formamide solution.The products of these reactions were resolved by electrophoresis throughseparate 20% denaturing polyacrylamide gels (19:1 cross-linked),containing 7 M urea, in a buffer of 45 mM Tris-Borate, pH 8.3, 1.4 mMEDTA. Following electrophoresis, the labeled reaction products werevisualized using the FMBIO-100 Image Analyzer (Hitachi), with theresulting imager scans shown in FIGS. 53A (5 min reactions) and 53B (30min. reactions).

[0570] In FIG. 53 the target RNA is seen very near the top of each lane,while the labeled probe and its cleavage products are seen just belowthe middle of each panel. The FMBIO-100 Image Analyzer was used toquantitate the fluorescence signal in the probe bands. In each panel,lane 1 contains products from reactions performed in the absence of acleavage agent, lane 2 contains products from reactions performed usingCleavase® A/G and lane 3 contains products from reactions performedusing DNAPTth.

[0571] Quantitation of the fluorescence signal in the probe bandsrevealed that after a 5 min. incubation, 12% or 300 fmole of the probewas cleaved by the Cleavase® A/G and 29% or 700 fmole was cleaved by theDNAPTth. After a 30 min. incubation, Cleavase® A/G had cleaved 32% ofthe probe molecules and DNAPTth had cleaved 70% of the probe molecules.(The images shown in FIGS. 53A and 53B were printed with the intensityadjusted to show the small amount of background from the RNAdegradation, so the bands containing strong signals are saturated andtherefore these images do not accurately reflect the differences inmeasured fluorescence) The data shown in FIG. 53 clearly shows that,under invasive cleavage conditions, RNA molecules are sufficientlystable to be detected as a target and that each RNA molecule can supportmany rounds of probe cleavage.

EXAMPLE 22 Titration of Target RNA in the Invader-Directed CleavageAssay

[0572] One of the primary benefits of the invader-directed cleavageassay as a means for detection of the presence of specific targetnucleic acids is the correlation between the amount of cleavage productgenerated in a set amount of time and the quantity of the nucleic acidof interest present in the reaction. The benefits of quantitativedetection of RNA sequences was discussed in Example 20. In this example,we demonstrate the quantitative nature of the detection assay throughthe use of various amounts of target starting material. In addition todemonstrating the correlation between the amounts of input target andoutput cleavage product, these data graphically show the degree to whichthe RNA target can be recycled in this assay

[0573] The RNA target used in these reactions was thefluorescein-labeled material described in Example 21 (i.e., SEQ IDNO:56). Because the efficiency of incorporation of thefluorescein-12-UTP by the T7 RNA polymerase was not known, theconcentration of the RNA was determined by measurement of absorbance at260 nm, not by fluorescence intensity. Each reaction comprised 5 pmolesof the fluorescein-labeled probe (SEQ ID NO:57) and 10 pmoles of theinvader oligonucleotide (SEQ ID NO:58) in a buffer of 10 mM MOPS, pH 7.5with 150 mM LiCl, 4 mM MnCl₂, 0.05% each Tween-20 and Nonidet-P40 and 39units of RNAsin® (Promega). The amount of target RNA was varied from 1to 100 fmoles, as indicated below. These components were combined,overlaid with ChillOut® evaporation barrier (MJ Research) and warmed to50° C.; the reactions were started by the addition of either 53 ng ofCleavase® A/G or 5 polymerase units of DNAPTth, to a final reactionvolume of 10 μl. After 30 minutes at 50° C., reactions were stopped bythe addition of 8 μl of 95% formamide, 10 mM EDTA and 0.02% methylviolet. The unreacted markers in lanes 1 and 2 were diluted in the sametotal volume (18 μl). The samples were heated to 90° C. for 1 minute and2.5 μl of each of these reactions were resolved by electrophoresisthrough a 20% denaturing polyacrylamide gel (19:1 cross link) with 7Murea in a buffer of 45 mM Tris-Borate, pH 8.3, 1.4 mM EDTA, and thelabeled reaction products were visualized using the FMBIO-100 ImageAnalyzer (Hitachi), with the resulting imager scans shown in FIG. 54.

[0574] In FIG. 54, lanes 1 and 2 show 5 pmoles of uncut probe and 500fmoles of untreated RNA, respectively. The probe is the very dark signalnear the middle of the panel, while the RNA is the thin line near thetop of the panel. These RNAs were transcribed with a 2% substitution offluorescein-12-UTP for natural UTP in the transcription reaction. Theresulting transcript contains 74 U residues, which would give an averageof 1.5 fluorescein labels per molecule. With one tenth the molar amountof RNA loaded in lane 2, the signal in lane 2 should be approximatelyone seventh (0.15×) the fluorescence intensity of the probe in lane 1.Measurements indicated that the intensity was closer to one fortieth,indicating an efficiency of label incorporation of approximately 17%.Because the RNA concentration was verified by A260 measurement this doesnot alter the experimental observations below, but it should be notedthat the signal from the RNA and the probes does not accurately reflectthe relative amounts in the reactions.

[0575] The reactions analyzed in lanes 3 through 7 contained 1, 5, 10,50 and 100 fmoles of target, respectively, with cleavage of the probeaccomplished by Cleavase® A/G. The reactions analyzed in lanes 8 through12 repeated the same array of target amounts, with cleavage of the probeaccomplished by DNAPTth. The boxes seen surrounding the product bandsshow the area of the scan in which the fluorescence was measured foreach reaction. The number of fluorescence units detected within each boxis indicated below each box; background florescence was also measured.

[0576] It can be seen by comparing the detected fluorescence in eachlane that the amount of product formed in these 30 minute reactions canbe correlated to the amount of target material. The accumulation ofproduct under these conditions is slightly enhanced when DNAPTth is usedas the cleavage agent, but the correlation with the amount of targetpresent remains. This demonstrates that the invader assay can be used asa means of measuring the amount of target RNA within a sample.

[0577] Comparison of the fluorescence intensity of the input RNA withthat of the cleaved product shows that the invader-directed cleavageassay creates signal in excess of the amount of target, so that thesignal visible as cleaved probe is far more intense than thatrepresenting the target RNA. This further confirms the results describedin Example >>, in which it was demonstrated that each RNA molecule couldbe used many times.

EXAMPLE 23 Detection of DNA by Charge Reversal

[0578] The detection of specific targets is achieved in theinvader-directed cleavage assay by the cleavage of the probeoligonucleotide. In addition to the methods described in the precedingexamples, the cleaved probe may be separated from the uncleaved probeusing the charge reversal technique described below. This novelseparation technique is related to the observation that positivelycharged adducts can affect the electrophoretic behavior of smalloligonucleotides because the charge of the adduct is significantrelative to charge of the whole complex. Observations of aberrantmobility due to charged adducts have been reported in the literature,but in all cases found, the applications pursued by other scientistshave involved making oligonucleotides larger by enzymatic extension. Asthe negatively charged nucleotides are added on, the positive influenceof the adduct is reduced to insignificance. As a result, the effects ofpositively charged adducts have been dismissed and have receivedinfinitesimal notice in the existing literature.

[0579] This observed effect is of particular utility in assays based onthe cleavage of DNA molecules. When an oligonucleotide is shortenedthrough the action of a Cleavase® enzyme or other cleavage agent, thepositive charge can be made to not only significantly reduce the netnegative charge, but to actually override it, effectively “flipping” thenet charge of the labeled entity. This reversal of charge allows theproducts of target-specific cleavage to be partitioned from uncleavedprobe by extremely simple means. For example, the products of cleavagecan be made to migrate towards a negative electrode placed at any pointin a reaction vessel, for focused detection without gel-basedelectrophoresis. When a slab gel is used, sample wells can be positionedin the center of the gel, so that the cleaved and uncleaved probes canbe observed to migrate in opposite directions. Alternatively, atraditional vertical gel can be used, but with the electrodes reversedrelative to usual DNA gels (i.e., the positive electrode at the top andthe negative electrode at the bottom) so that the cleaved moleculesenter the gel, while the uncleaved disperse into the upper reservoir ofelectrophoresis buffer.

[0580] An additional benefit of this type of readout is that theabsolute nature of the partition of products from substrates means thatan abundance of uncleaved probe can be supplied to drive thehybridization step of the probe-based assay, yet the unconsumed probecan be subtracted from the result to reduce background.

[0581] Through the use of multiple positively charged adducts, syntheticmolecules can be constructed with sufficient modification that thenormally negatively charged strand is made nearly neutral. When soconstructed, the presence or absence of a single phosphate group canmean the difference between a net negative or a net positive charge.This observation has particular utility when one objective is todiscriminate between enzymatically generated fragments of DNA, whichlack a 3′ phosphate, and the products of thermal degradation, whichretain a 3′ phosphate (and thus two additional negative charges).

[0582] a) Characterization of the Products of Thermal Breakage of DNAOligonucleotides

[0583] Thermal degradation of DNA probes results in high backgroundwhich can obscure signals generated by specific enzymatic cleavage,decreasing the signal-to-noise ratio. To better understand the nature ofDNA thermal degradation products, we incubated the 5′tetrachloro-fluorescein (TET)-labeled oligonucleotides 78 (SEQ ID NO:59)and 79 (SEQ ID NO:60) (100 pmole each) in 50 μl 10 mM NaCO₃ (pH 10.6),50 mM NaCl at 90° C. for 4 hours. To prevent evaporation of the samples,the reaction mixture was overlaid with 50 μl of ChillOut® 14 liquid wax(MJ Research). The reactions were then divided in two equal aliquots (Aand B). Aliquot A was mixed with 25 μl of methyl violet loading bufferand Aliquot B was dephosphorylated by addition of 2.5 μl of 100 mM MgCl₂and 1 μl of 1 unit/μl Calf Intestinal Alkaline Phosphatase (CIAP)(Promega), with incubation at 37° C. for 30 min. after which 25 μl ofmethyl violet loading buffer was added. One microliter of each samplewas resolved by electrophoresis through a 12% polyacrylamide denaturinggel and imaged as described in Example 21; a 585 nm filter was used withthe FMBIO Image Analyzer. The resulting imager scan is shown in FIG. 55.In FIG. 55, lanes 1-3 contain the TET-labeled oligonucleotide 78 andlanes 4-6 contain the TET-labeled oligonucleotides 79. Lanes 1 and 4contain products of reactions which were not heat treated. Lanes 2 and 5contain products from reactions which were heat treated and lanes 3 and6 contain products from reactions which were heat treated and subjectedto phosphatase treatment.

[0584] As shown in FIG. 55, heat treatment causes significant breakdownof the 5′-TET-labeled DNA, generating a ladder of degradation products(FIG. 55, lanes 2, 3, 5 and 6). Band intensities correlate with purineand pyrimidine base positioning in the oligonucleotide sequences,indicating that backbone hydrolysis may occur through formation ofabasic intermediate products that have faster rates for purines then forpyrimidines [Lindahl and Karlstrom (1973) Biochem. 12:5151].

[0585] Dephosphorylation decreases the mobility of all productsgenerated by the thermal degradation process, with the most pronouncedeffect observed for the shorter products (FIG. 55, lanes 3 and 6). Thisdemonstrates that thermally degraded products possess a 3′ end terminalphosphoryl group which can be removed by dephosphorylation with CIAP.Removal of the phosphoryl group decreases the overall negative charge by2. Therefore, shorter products which have a small number of negativecharges are influenced to a greater degree upon the removal of twocharges. This leads to a larger mobility shift in the shorter productsthan that observed for the larger species.

[0586] The fact that the majority of thermally degraded DNA productscontain 3′ end phosphate groups and Cleavase® enzyme-generated productsdo not allowed the development of simple isolation methods for productsgenerated in the invader-directed cleavage assay. The extra two chargesfound in thermal breakdown products do not exist in the specificcleavage products. Therefore, if one designs assays that producespecific products which contain a net positive charge of one or two,then similar thermal breakdown products will either be negative orneutral. The difference can be used to isolate specific products byreverse charge methods as shown below.

[0587] b) Dephosphorylation of Short Amino-Modified Oligonucleotides canReverse the Net Charge of the Labeled Product

[0588] To demonstrate how oligonucleotides can be transformed from netnegative to net positively charged compounds, the four shortamino-modified oligonucleotides labeled 70, 74, 75 and 76 and shown inFIGS. 56-58 were synthesized (FIG. 56 shows both oligonucleotides 70 and74). All four modified oligonucleotides possess Cy-3 dyes positioned atthe 5′-end which individually are positively charged under reaction andisolation conditions described in this example. Compounds 70 and 74contain two amino modified thymidines that, under reaction conditions,display positively charged R-NH₃+groups attached at the C5 positionthrough a C₁₀ or C₆ linker, respectively. Because compounds 70 and 74are 3′-end phosphorylated, they consist of four negative charges andthree positive charges. Compound 75 differs from 74 in that the internalC₆ amino modified thymidine phosphate in 74 is replaced by a thymidinemethyl phosphonate. The phosphonate backbone is uncharged and so thereare a total of three negative charges on compound 75. This givescompound 75 a net negative one charge. Compound 76 differs from 70 inthat the internal amino modified thymidine is replaced by an internalcytosine phosphonate. The pK_(a) of the N3 nitrogen of cytosine can befrom 4 to 7. Thus, the net charges of this compound, can be from −1 to 0depending on the pH of the solution. For the simplicity of analysis,each group is assigned a whole number of charges, although it isrealized that, depending on the pK_(a) of each chemical group andambient pH, a real charge may differ from the whole number assigned. Itis assumed that this difference is not significant over the range of pHsused in the enzymatic reactions studied here.

[0589] Dephosphorylation of these compounds, or the removal of the 3′end terminal phosphoryl group, results in elimination of two negativecharges and generates products that have a net positive charge of one.In this experiment, the method of isoelectric focusing (IEF) was used todemonstrate a change from one negative to one positive net charge forthe described substrates during dephosphorylation.

[0590] Substrates 70, 74, 75 and 76 were synthesized by standardphosphoramidite chemistries and deprotected for 24 hours at 22° C. in 14M aqueous ammonium hydroxide solution, after which the solvent wasremoved in vacuo. The dried powders were resuspended in 200 μl of H20and filtered through 0.2 μm filters. The concentration of the stocksolutions was estimated by UV-absorbance at 261 nm of samples diluted200-fold in H₂O using a spectrophotometer (Spectronic Genesys 2, MiltonRoy, Rochester, N.Y.).

[0591] Dephosphorylation of compounds 70 and 74, 75 and 76 wasaccomplished by treating 10 μl of the crude stock solutions (ranging inconcentration from approximately 0.5 to 2 mM) with 2 units of CIAP in100 μl of CIAP buffer (Promega) at 37° C. for 1 hour. The reactions werethen heated to 75° C. for 15 min. in order to inactivate the CIAP. Forclarity, dephosphorylated compounds are designated ‘dp’. For example,after dephosphorylation, substrate 70 becomes 70dp.

[0592] To prepare samples for IEF experiments, the concentration of thestock solutions of substrate and dephosphorylated product were adjustedto a uniform absorbance of 8.5×10⁻³ at 532 nm by dilutuion with water.Two microliters of each sample were analyzed by IEF using a PhastSystemelectrophoresis unit (Pharmacia) and PhastGel IEF 3-9 media (Pharmacia)according to the manufacturer's protocol. Separation was performed at15° C. with the following program: pre-run; 2,000 V, 2.5 mA, 3.5 W, 75Vh; load; 200 V, 2.5 mA, 3.5 W, 15 Vh; Vh; 2,000 V; 2.5 mA; 3.5 W, 130Vh. After separation, samples were visualized by using the FMBIO ImageAnalyzer (Hitachi) fitted with a 585 nm filter. The resulting imagerscan is shown in FIG. 59.

[0593]FIG. 59 shows results of IEF separation of substrates 70, 74, 75and 76 and their dephosphorylated products. The arrow labeled “SampleLoading Position” indicates a loading line, the ‘+’ sign shows theposition of the positive electrode and the ‘−’ sign indicates theposition of the negative electrode.

[0594] The results shown in FIG. 59 demonstrate that substrates 70, 74,75 and 76 migrated toward the positive electrode, while thedephosphorylated products 70dp, 74dp, 75dp and 76dp migrated towardnegative electrode. The observed differences in mobility direction wasin accord with predicted net charge of the substrates (minus one) andthe products (plus one). Small perturbations in the mobilities of thephosphorylated compounds indicate that the overall pIs vary. This wasalso true for the dephosphorylated compounds. The presence of thecytosine in 76dp, for instance, moved this compound further toward thenegative electrode which was indicative of a higher overall pI relativeto the other dephosphorylated compounds. It is important to note thatadditional positive charges can be obtained by using a combination ofnatural amino modified bases (70dp and 74dp) along with unchargedmethylphosphonate bridges (products 75dp and 76dp).

[0595] The results shown above demonstrate that the removal of a singlephosphate group can flip the net charge of an oligonucleotide to causereversal in an electric field, allowing easy separation of products, andthat the precise base composition of the oligonucleotides affectabsolute mobility but not the charge-flipping effect.

EXAMPLE 23 Detection of Specific Cleavage Products in theInvader-Directed Cleavage Reaction by Charge Reversal

[0596] In this example the ability to isolate products generated in theinvader-directed cleavage assay from all other nucleic acids present inthe reaction cocktail was demonstrated using charge reversal. Thisexperiment utilized the following Cy3-labeled oligonucleotide:5′-Cy3-AminoT-AminoT-CTTTTCACCAGCGAGACGGG-3′ (SEQ ID NO:61; termed“oligo 61”). Oligo 61 was designed to release upon cleavage a netpositively charged labeled product. To test whether or not a netpositively charged 5′-end labeled product would be recognized by theCleavase® enzymes in the invader-directed cleavage assay format, probeoligo 61 (SEQ ID NO:61) and invading oligonucleotide 67 (SEQ ID NO:62)were chemically synthesized on a DNA synthesizer (ABI 391) usingstandard phosphoramidite chemistries and reagents obtained from GlenResearch (Sterling, Va.).

[0597] Each assay reaction comprised 100 fmoles of M13mp18 singlestranded DNA, 10 pmoles each of the probe (SEQ ID NO:61) and invader(SEQ ID NO:62) oligonucleotides, and 20 units of Cleavase® A/G in a 10μl solution of 10 mM MOPS, pH 7.4 with 100 mM KCl. Samples were overlaidwith mineral oil to prevent evaporation. The samples were brought toeither 50° C., 55° C., 60° C., or 65° C. and cleavage was initiated bythe addition of 1 μl of 40 mM MnCl₂. Reactions were allowed to proceedfor 25 minutes and then were terminated by the addition of 10 μl of 95%formamide containing 20 mM EDTA and 0.02% methyl violet. The negativecontrol experiment lacked the target M13mp18 and was run at 60° C. Fivemicroliters of each reaction were loaded into separate wells of a 20%denaturing polyacrylamide gel (cross-linked 29:1) with 8 M urea in abuffer containing 45 mM Tris-Borate (pH 8.3) and 1.4 mM EDTA. Anelectric field of 20 watts was applied for 30 minutes, with theelectrodes oriented as indicated in FIG. 60B (i.e., in reverseorientation). The products of these reactions were visualized using theFMBIO fluorescence imager and the resulting imager scan is shown in FIG.60B.

[0598]FIG. 60A provides a schematic illustration showing an alignment ofthe invader (SEQ ID NO:61) and probe (SEQ ID NO:62) along the targetM13mp18 DNA; only 53 bases of the M13mp18 sequence is shown (SEQ IDNO:63). The sequence of the inavder oligonucleotide is displayed underthe M13mp18 target and an arrow is used above the M13mp18 sequence toindicate the position of the invader relative to the probe and target.As shown in FIG. 60A, the invader and probe oligonucleotides share a 2base region of overlap.

[0599] In FIG. 60B, lanes 1-6 contain reactions peformed at 50° C., 55°C., 60° C., and 65° C., respectively; lane 5 contained the controlreaction (lacking target). In FIG. 60B, the products of cleavage areseen as dark bands in the upper half of the panel; the faint lower bandseen appears in proportion to the amount of primary product producedand, while not limiting the invetion to a particular mechanism, mayrepresent cleavage one nucleotide into the duplex. The uncleaved probedoes not enter the gel and is thus not visible. The control lane showedno detectable signal over background (lane 5). As expected in aninvasive cleavage reaction, the rate of accumulation of specificcleavage product was temperature-dependent. Using these particularoligonucleotides and target, the fastest rate of accumulation of productwas observed at 55° C. (lane 2) and very little product observed at 65°C. (lane 4).

[0600] When incubated for extended periods at high temperature, DNAprobes can break non-specifically (i.e., suffer thermal degradation) andthe resulting fragments contribute an interfering background to theanalysis. The products of such thermal breakdown are distributed fromsingle-nucleotides up to the full length probe. In this experiment, theability of charge based separation of cleavage products (i.e., chargereversal) would allow the sensitve separation of the specific productsof target-dependent cleavage from probe fragments generated by thermaldegradation was examined.

[0601] To test the sensitivity limit of this detection method, thetarget M13mp18 DNA was serially diluted ten fold over than range of 1fmole to 1 amole. The invader and probe oligonucleotides were thosedecribed above (i.e., SEQ ID NOS:61 and 62). The invasive cleavagereactions were run as described above with the following modifications:the reactions were performed at 55° C., 250 mM or 100 mM KGlu was usedin place of the 100 mM KCl and only 1 pmole of the invaderoligonucleotide was added. The reactions were initiated as describedabove and allowed to progress for 12.5 hours. A negative controlreaction which lacked added M13m18 target DNA was also run. Thereactions were terminated by the addition of 10 μl of 95% formamidecontaining 20 mM EDTA and 0.02% methyl violet, and 5 μl of thesemixtures were electrophoresed and visualized as described above. Theresulting imager scan is shown in FIG. 61.

[0602] In FIG. 61, lane 1 contains the regative control; lanes 2-5contain reactions performed using 100 mM KGlu; lanes 6-9 containreactions performed using 250 mM KGlu. The reactions resolved in lanes 2and 6 contained 1 fmole of target DNA; those in lanes 3 and 7 contained100 amole of target; those in lanes 4 and 8 contained 10 amole of targetand those in lanes 5 and 9 contained 1 amole of target. The resultsshown in FIG. 61 demonstrate that the detection limit using chargereversal to detect the production of specific cleavage products in aninvasive cleavage reaction is at or below 1 attomole or approximately6.02×10⁵ target molecules. No detectable signal was observed in thecontrol lane, which indicates that non-specific hydrolysis or otherbreakdown products do not migrate in the same direction asenzyme-specific cleavage products. The excitation and emission maximafor Cy3 are 554 and 568, respectively, while the FMBIO Imager Analyzerexcites at 532 and detects at 585. Therefore, the limit of detection ofspecific cleavage products can be improved by the use of more closelymatched excitation source and detection filters.

EXAMPLE 24 Devices and Methods for the Separation and Detection ofCharged Reaction Products

[0603] This example is directed at methods and devices for isolating andconcentrating specific reaction products produced by enzymatic reactionsconducted in solution whereby the reactions generate charged productsfrom either a charge neutral substrate or a substrate bearing theopposite charge borne by the specific reaction product. The methods anddevices of this example allow isolation of, for example, the productsgenerated by the invader-directed cleavage assay of the presentinvention.

[0604] The methods and devices of this example are based on theprinciple that when an electric field is applied to a solution ofcharged molecules, the migration of the molecules toward the electrodeof the opposite charge occurs very rapidly. If a matrix or otherinhibitory material is introduced between the charged molecules and theelectrode of opposite charge such that this rapid migration isdramatically slowed, the first molecules to reach the matrix will benearly stopped, thus allowing the lagging molecules to catch up. In thisway a dispersed population of charged molecules in solution can beeffectively concentrated into a smaller volume. By tagging the moleculeswith a detectable moiety (e.g., a fluorescent dye), detection isfacilitated by both the concentration and the localization of theanalytes. This example illustrates two embodiments of devicescontemplated by the present invention; of course, variations of thesedevices will be apparent to those skilled in the art and are within thespirit and scope of the present invention.

[0605]FIG. 62 depicts one embodiment of a device for concentrating thepositively-charged products generated using the methods of the presentinvention. As shown in FIG. 62, the device comprises a reaction tube(10) which contains the reaction solution (11). One end of each of twothin capillaries (or other tubes with a hollow core) (13A and 13B) aresubmerged in the reaction solution (11). The capillaries (13A and 13B)may be suspended in the reaction solution (11) such that they are not incontact with the reaction tube itself; one appropriate method ofsuspending the capillaries is to hold them in place with clamps (notshown). Alternatively, the capillaries may be suspended in the reactionsolution (11) such that they are in contact with the reaction tubeitself. Suitable capillaries include glass capillary tubes commonlyavailable from scientific supply companies (e.g., Fisher Scientific orVWR Scientific) or from medical supply houses that carry materials forblood drawing and analysis. Though the present invention is not limitedto capillaries of any particular inner diameter, tubes with innerdiameters of up to about ⅛ inch (approximately 3 mm) are particularlypreferred for use with the present invention; for example Kimble No.73811-99 tubes (VWR Scientific) have an inner diameter of 1.1 mm and area suitable type of capillary tube. Although the capillaries of thedevice are commonly composed of glass, any nonconductive tubularmaterial, either rigid or flexible, that can contain either a conductivematerial or a trapping material is suitable for use in the presentinvention. One example of a suitable flexible tube is Tygon® clearplastic tubing (Part No. R3603; inner diameter={fraction (1/16)} inch;outer diameter=⅛ inch).

[0606] As illustrated in FIG. 62, capillary 13A is connected to thepositive electrode of a power supply (20) (e.g., a controllable powersupply available through the laboratory suppliers listed above orthrough electronics supply houses like Radio Shack) and capillary 13B isconnected to the negative electrode of the power supply (20). Capillary13B is filled with a trapping material (14) capable of trapping thepositively-charged reaction products by allowing minimal migration ofproducts that have entered the trapping material (14). Suitable trappingmaterials include, but are not limited to, high percentage (e.g., about20%) acrylamide polymerized in a high salt buffer (0.5 M or highersodium acetate or similar salt); such a high percentage polyacrylamidematrix dramatically slows the migration of the positively-chargedreaction products. Alternatively, the trapping material may comprise asolid, negatively-charged matrix, such as negatively-charged latexbeads, that can bind the incoming positively-charged products. It shouldbe noted that any amount of trapping material (14) capable of inhibitingany concentrating the positively-charged reaction products may be used.Thus, while the capillary 13B in FIG. 62 only contains trapping materialin the lower, submerged portion of the tube, the trapping material (14)can be present in the entire capillary (13B); similarly, less trappingmaterial (14) could be present than that shown in FIG. 62 because thepositively-charged reaction products generally accumulate within a verysmall portion of the bottom of the capillary (13B). The amount oftrapping material need only be sufficient to make contact with thereaction solution (11) and have the capacity to collect the reactionproducts. When capillary 13B is not completely filled with the trappingmaterial, the remaining space is filled with any conductive material(15); suitable conductive materials are discussed below.

[0607] By comparison, the capillary (13A) connected to the positiveelectrode of the power supply 20 may be filled with any conductivematerial (15; indicated by the hatched lines in FIG. 62). This may bethe sample reaction buffer (e.g., 10 mM MOPS, pH 7.5 with 150 mM LiCl, 4mM MnCl₂), a standard electrophoresis buffer (e.g., 45 mM Tris-Borate,pH 8.3, 1.4 mM EDTA), or the reaction solution (11) itself. Theconductive material (15) is frequently a liquid, but a semi-solidmaterial (e.g., a gel) or other suitable material might be easier to useand is within the scope of the present invention. Moreover, thattrapping material used in the other capillary (i.e., capillary 13B) mayalso be used as the conductive material. Conversely, it should be notedthat the same conductive material used in the capillary (13A) attachedto the positive electrode may also be used in capillary 13B to fill thespace above the region containing the trapping material (14) (see FIG.62).

[0608] The top end of each of the capillaries (13A and 13B) is connectedto the appropriate electrode of the power supply (20) by electrode wire(18) or other suitable material. Fine platinum wire (e.g., 0.1 to 0.4mm, Aesar Johnson Matthey, Ward Hill, Mass.) is commonly used asconductive wire because it does not corrode under electrophoresisconditions. The electrode wire (18) can be attached to the capillaries(13A and 13B) by a nonconductive adhesive (not shown), such as thesilicone adhesives that are commonly sold in hardware stores for sealingplumbing fixtures. If the capillaries are constructed of a flexiblematerial, the electrode wire (18) can be secured with a small hose clampor constricting wire (not shown) to compress the opening of thecapillaries around the electrode wire. If the conducting material (15)is a gel, an electrode wire (18) can be embedded directly in the gelwithin the capillary.

[0609] The cleavage reaction is assembled in the reaction tube (10) andallowed to proceed therein as described in proceeding examples (e.g,Examples 22-23). Though not limited to any particular volume of reactionsolution (11), a preferred volume is less than 10 ml and more preferablyless than 0.1 ml. The volume need only be sufficient to permit contactwith both capillaries. After the cleavage reaction is completed, anelectric field is applied to the capillaries by turning on the powersource (20). As a result, the positively-charged products generated inthe course of the invader-directed cleavage reaction which employs anoligonucleotide, which when cleaved, generates a positively chargedfragment (described in Ex. 23) but when uncleaved bears a net negativecharge, migrate to the negative capillary, where their migration isslowed or stopped by the trapping material (14), and thenegatively-charged uncut and thermally degraded probe molecules migratetoward the positive electrode. Through the use of this or a similardevice, the positively-charged products of the invasive cleavagereaction are separated from the other material (i.e., uncut andthermally degraded probe) and concentrated from a large volume.Concentration of the product in a small amount of trapping material (14)allows for simplicity of detection, with a much higher signal-to-noiseratio than possible with detection in the original reaction volume.Because the concentrated product is labelled with a detectable moietylike a fluorescent dye, a commercially-available fluorescent platereader (not shown) can be used to ascertain the amount of product.Suitable plate readers include both top and bottom laser readers.Capillary 13B can be positioned with the reaction tube (10) at anydesired position so as to accommodate use with either a top or a bottomplate reading device.

[0610] In the alternative embodiment of the present invention depictedin FIG. 63, the procedure described above is accomplished by utilizingonly a single capillary (13B). The capillary (13B) contains the trappingmaterial (14) described above and is connected to an electrode wire(18), which in turn is attached to the negative electrode of a powersupply (20). The reaction tube (10) has an electrode (25) embedded intoits surface such that one surface of the electrode is exposed to theinterior of the reaction tube (10) and another surface is exposed to theexterior of the reaction tube. The surface of the electrode (25) on theexterior of the reaction tube is in contact with a conductive surface(26) connected to the positive electrode of the power supply (20)through an electrode wire (18). Variations of the arrangement depictedin FIG. 63 are also contemplated by the present invention. For example,the electrode (25) may be in contact with the reaction solution (11)through the use of a small hole in the reaction tube (10); furthermore,the electrode wire (18) can be directly attached to the electrode wire(18), thereby eliminating the conductive surface (26).

[0611] As indicated in FIG. 63, the electrode (25) is embedded in thebottom of a reaction tube (10) such that one or more reaction tubes maybe set on the conductive surface (26). This conductive surface couldserve as a negative electrode for multiple reaction tubes; such asurface with appropriate contacts could be applied through the use ofmetal foils (e.g., copper or platinum, Aesar Johnson Matthey, Ward Hill,Mass.) in much the same way contacts are applied to circuit boards.Because such a surface contact would not be exposed to the reactionsample directly, less expensive metals, such as the copper could be usedto make the electrical connections.

[0612] The above devices and methods are not limited to separation andconcentration of positively charged oligonucleotides. As will beapparent to those skilled in the art, negatively charged reactionproducts may be separated from neutral or positively charged reactantsusing the above device and methods with the exception that capillary 13Bis attached to the positive electrode of the power supply (20) andcapillary 13A or alternatively, electrode 25, is attached to thenegative electrode of the power supply (20).

EXAMPLE 25 Primer-Directed and Primer Independent Cleavage Occur at theSame Site when the Primer Extends to the 3′ Side of a Mismatched“Bubble” in the Downstream Duplex

[0613] As discussed above in Example 1, the presence of a primerupstream of a bifurcated duplex can influence the site of cleavage, andthe existence of a gap between the 3′ end of the primer and the base ofthe duplex can cause a shift of the cleavage site up the unpaired 5′ armof the structure (see also Lyamichev et al., supra and U.S. Pat. No.5,422,253). The resulting non-invasive shift of the cleavage site inresponse to a primer is demonstrated in FIGS. 9, 10 and 11, in which theprimer used left a 4-nucleotide gap (relative to the base of theduplex). In FIGS. 9-11, all of the “primer-directed” cleavage reactionsyielded a 21 nucleotide product, while the primer-independent cleavagereactions yielded a 25 nucleotide product. The site of cleavage obtainedwhen the primer was extended to the base of the duplex, leaving no gapwas examined. The results are shown in FIG. 64 (FIG. 64 is areproduction of FIG. 2C in Lyamichev et al. These data were derived fromthe cleavage of the structure shown in FIG. 6, as described inExample 1. Unless otherwise specified, the cleavage reactions comprised0.01 pmoles of heat-denatured, end-labeled hairpin DNA (with theunlabeled complementary strand also present), I pmole primer[complementary to the 3′ arm shown in FIG. 6 and having the sequence:5′-GAAT TCGATTTAGGTGACACTATAGAATACA (SEQ ID NO:64)] and 0.5 units ofDNAPTaq (estimated to be 0.026 pmoles) in a total volume of 10 μl of 10mM Tris-Cl, pH 8.5, and 1.5 mM MgCl₂ and 50 mM KCl. The primer wasomitted from the reaction shown in the first lane of FIG. 64 andincluded in lane 2. These reactions were incubated at 55° C. for 10minutes. Reactions were initiated at the final reaction temperature bythe addition of either the MgCl₂ or enzyme. Reactions were stopped attheir incubation temperatures by the addition of 8 μl of 95% formamidewith 20 mM EDTA and 0.05% marker dyes.

[0614]FIG. 64 is an autoradiogram that indicates the effects on the siteof cleavage of a bifurcated duplex structure in the presence of a primerthat extends to the base of the hairpin duplex. The size of the releasedcleavage product is shown to the left (i.e., 25 nucleotides). Adideoxynucleotide sequencing ladder of the cleavage substrate is shownon the right as a marker (lanes 3-6).

[0615] These data show that the presence of a primer that is adjacent toa downstream duplex (lane 2) produces cleavage at the same site as seenin reactions performed in the absence of the primer (lane 1) (see FIGS.9A and B, 10B and 11A for additional comparisons). When the 3′ terminalnucleotides of the upstream oligonucleotide can base pair to thetemplate strand but are not homologous to the displaced strand in theregion immediately upstream of the cleavage site (i.e., when theupstream oligonucleotide is opening up a “bubble” in the duplex), thesite to which cleavage is apparently shifted is not wholly dependent onthe presence of an upstream oligonucleotide.

[0616] As discussed above in the Background section and in Table 1, therequirement that two independent sequences be recognized in an assayprovides a highly desirable level of specificity. In the invasivecleavage reactions of the present invention, the invader and probeoligonucleotides must hybridize to the target nucleic acid with thecorrect orientation and spacing to enable the production of the correctcleavage product. When the distinctive pattern of cleavage is notdependent on the successful alignment of both oligonucleotides in thedetection system these advantages of independent recognition are lost.

EXAMPLE 26 Invasive Cleavage and Primer-Directed Cleavage when there isOnly Partial Homology in the “X” Overlap Region

[0617] While not limiting the present invention to any particularmechanism, invasive cleavage occurs when the site of cleavage is shiftedto a site within the duplex formed between the probe and the targetnucleic acid in a manner that is dependent on the presence of anupstream oligonucleotide which shares a region of overlap with thedownstream probe oligonucleotide. In some instances, the 5′ region ofthe downstream oligonucleotide may not be completely complementary tothe target nucleic acid. In these instances, cleavage of the probe mayoccur at an internal site within the probe even in the absence of anupstream oligonucleotide (in contrast to the base-by-base nibbling seenwhen a fully paired probe is used without an invader). Invasive cleavageis characterized by an apparent shifting of cleavage to a site within adownstream duplex that is dependent on the presence of the invaderoligonucleotide.

[0618] A comparision between invasive cleavage and primer-directedcleavagem may be illustrated by comparing the expected cleavage sites ofa set of probe oligonucleotides having decreasing degrees ofcomplementarity to the target strand in the 5′ region of the probe(i.e., the region that overlaps with the invader). A simple test,similar to that performed on the hairpin substrate above (Ex. 25), canbe performed to compare invasive cleavage with the non-invasiveprimer-directed cleavage described above. Such a set of testoligonucleotides is diagrammed in FIG. 65. The structures shown in FIG.65 are grouped in pairs, labeled “a”, “b”, “c”, and “d”. Each pair hasthe same probe sequence annealed to the target strand (SEQ ID NO:65),but the top structure of each pair is drawn without an upstreamoligonucleotide, while the bottom structure includes thisoligonucleotide (SEQ ID NO:66). The sequences of the probes shown inFIGS. 64a-64 d are listed in SEQ ID NOS:43, 67, 68 and 69, respectively.Probable sites of cleavage are indicated by the black arrowheads. (It isnoted that the precise site of cleavage on each of these structures mayvary depending on the choice of cleavage agent and other experimentalvariables. These particular sites are provided for illustrative purposesonly.) To conduct this test, the site of cleavage of each probe isdetermined both in the presence and the absence of the upstreamoligonucleotide, in reaction conditions such as those described inExample 19. The products of each pair of reactions are then be comparedto determine whether the fragment released from the 5′ end of the probeincreases in size when the upstream oligonucleotide is included in thereaction.

[0619] The arrangement shown in FIG. 65a, in which the probe molecule iscompletely complementary to the target strand, is similar to that shownin FIG. 32. Treatment of the top structure with the 5′ nuclease of a DNApolymerase would cause exonucleolytic nibbling of the probe (i.e., inthe absence of the upstream oligonucleotide). In contrast, inclusion ofan invader oligonucleotide would cause a distinctive cleavage shiftsimilar, to those observed in FIG. 33.

[0620] The arrangements shown in FIGS. 65b and 65 c have some amount ofunpaired sequence at the 5′ terminus of the probe (3 and 5 bases,respectively). These small 5′ arms are suitable cleavage substrate forthe 5′ nucleases and would be cleaved within 2 nucleotide's of thejunction between the single stranded region and the duplex. In thesearrangements, the 3′ end of the upstream oligonucleotide shares identitywith a portion of the 5′ region of the probe which is complementary tothe target sequence (that is the 3′ end of the invader has to competefor binding to the target with a portion of the 5′ end of the probe).Therefore, when the upstream oligonucleotide is included it is thoughtto mediate a shift in the site of cleavage into the downstream duplex(although the present invention is not limited to any particularmechanism of action), and this would, therefore, constitute invasivecleavage. If the extreme 5′ nucleotides of the unpaired region of theprobe were able to hybridize to the target strand, the cleavage site inthe absence of the invader might change but the addition of the invaderoligonucleotide would still shift the cleavage site to the properposition.

[0621] Finally, in the arrangement shown in FIG. 65d, the probe andupstream oligonucleotides share no significant regions of homology, andthe presence of the upstream oligonucleotide would not compete forbinding to the target with the probe. Cleavage of the structures shownin FIG. 64d would occur at the same site with or without the upstreamoligonucleotide, and is thus would not constitute invasive cleavage.

[0622] By examining any upstream oligonucleotide/probe pair in this way,it can easily be determined whether the resulting cleavage is invasiveor merely primer-directed. Such analysis is particularly useful when theprobe is not fully complementary to the target nucleic acid, so that theexpected result may not be obvious by simple inspection of thesequences.

[0623] From the above it is clear that the invention provides reagentsand methods to permit the detection and characterization of nucleic acidsequences and variations in nucleic acid sequences. The invader-directedcleavage reaction of the present invention provides an ideal directdetection method that combines the advantages of the direct detectionassays (e.g., easy quantification and minimal risk of carry-overcontamination) with the specificity provided by a dual oligonucleotidehybridization assay.

1 48 2506 base pairs nucleic acid double linear DNA (genomic) 1ATGAGGGGGA TGCTGCCCCT CTTTGAGCCC AAGGGCCGGG TCCTCCTGGT GGACGGCCAC 60CACCTGGCCT ACCGCACCTT CCACGCCCTG AAGGGCCTCA CCACCAGCCG GGGGGAGCCG 120GTGCAGGCGG TCTACGGCTT CGCCAAGAGC CTCCTCAAGG CCCTCAAGGA GGACGGGGAC 180GCGGTGATCG TGGTCTTTGA CGCCAAGGCC CCCTCCTTCC GCCACGAGGC CTACGGGGGG 240TACAAGGCGG GCCGGGCCCC CACGCCGGAG GACTTTCCCC GGCAACTCGC CCTCATCAAG 300GAGCTGGTGG ACCTCCTGGG GCTGGCGCGC CTCGAGGTCC CGGGCTACGA GGCGGACGAC 360GTCCTGGCCA GCCTGGCCAA GAAGGCGGAA AAGGAGGGCT ACGAGGTCCG CATCCTCACC 420GCCGACAAAG ACCTTTACCA GCTCCTTTCC GACCGCATCC ACGTCCTCCA CCCCGAGGGG 480TACCTCATCA CCCCGGCCTG GCTTTGGGAA AAGTACGGCC TGAGGCCCGA CCAGTGGGCC 540GACTACCGGG CCCTGACCGG GGACGAGTCC GACAACCTTC CCGGGGTCAA GGGCATCGGG 600GAGAAGACGG CGAGGAAGCT TCTGGAGGAG TGGGGGAGCC TGGAAGCCCT CCTCAAGAAC 660CTGGACCGGC TGAAGCCCGC CATCCGGGAG AAGATCCTGG CCCACATGGA CGATCTGAAG 720CTCTCCTGGG ACCTGGCCAA GGTGCGCACC GACCTGCCCC TGGAGGTGGA CTTCGCCAAA 780AGGCGGGAGC CCGACCGGGA GAGGCTTAGG GCCTTTCTGG AGAGGCTTGA GTTTGGCAGC 840CTCCTCCACG AGTTCGGCCT TCTGGAAAGC CCCAAGGCCC TGGAGGAGGC CCCCTGGCCC 900CCGCCGGAAG GGGCCTTCGT GGGCTTTGTG CTTTCCCGCA AGGAGCCCAT GTGGGCCGAT 960CTTCTGGCCC TGGCCGCCGC CAGGGGGGGC CGGGTCCACC GGGCCCCCGA GCCTTATAAA 1020GCCCTCAGGG ACCTGAAGGA GGCGCGGGGG CTTCTCGCCA AAGACCTGAG CGTTCTGGCC 1080CTGAGGGAAG GCCTTGGCCT CCCGCCCGGC GACGACCCCA TGCTCCTCGC CTACCTCCTG 1140GACCCTTCCA ACACCACCCC CGAGGGGGTG GCCCGGCGCT ACGGCGGGGA GTGGACGGAG 1200GAGGCGGGGG AGCGGGCCGC CCTTTCCGAG AGGCTCTTCG CCAACCTGTG GGGGAGGCTT 1260GAGGGGGAGG AGAGGCTCCT TTGGCTTTAC CGGGAGGTGG AGAGGCCCCT TTCCGCTGTC 1320CTGGCCCACA TGGAGGCCAC GGGGGTGCGC CTGGACGTGG CCTATCTCAG GGCCTTGTCC 1380CTGGAGGTGG CCGAGGAGAT CGCCCGCCTC GAGGCCGAGG TCTTCCGCCT GGCCGGCCAC 1440CCCTTCAACC TCAACTCCCG GGACCAGCTG GAAAGGGTCC TCTTTGACGA GCTAGGGCTT 1500CCCGCCATCG GCAAGACGGA GAAGACCGGC AAGCGCTCCA CCAGCGCCGC CGTCCTGGAG 1560GCCCTCCGCG AGGCCCACCC CATCGTGGAG AAGATCCTGC AGTACCGGGA GCTCACCAAG 1620CTGAAGAGCA CCTACATTGA CCCCTTGCCG GACCTCATCC ACCCCAGGAC GGGCCGCCTC 1680CACACCCGCT TCAACCAGAC GGCCACGGCC ACGGGCAGGC TAAGTAGCTC CGATCCCAAC 1740CTCCAGAACA TCCCCGTCCG CACCCCGCTT GGGCAGAGGA TCCGCCGGGC CTTCATCGCC 1800GAGGAGGGGT GGCTATTGGT GGCCCTGGAC TATAGCCAGA TAGAGCTCAG GGTGCTGGCC 1860CACCTCTCCG GCGACGAGAA CCTGATCCGG GTCTTCCAGG AGGGGCGGGA CATCCACACG 1920GAGACCGCCA GCTGGATGTT CGGCGTCCCC CGGGAGGCCG TGGACCCCCT GATGCGCCGG 1980GCGGCCAAGA CCATCAACTT CGGGGTCCTC TACGGCATGT CGGCCCACCG CCTCTCCCAG 2040GAGCTAGCCA TCCCTTACGA GGAGGCCCAG GCCTTCATTG AGCGCTACTT TCAGAGCTTC 2100CCCAAGGTGC GGGCCTGGAT TGAGAAGACC CTGGAGGAGG GCAGGAGGCG GGGGTACGTG 2160GAGACCCTCT TCGGCCGCCG CCGCTACGTG CCAGACCTAG AGGCCCGGGT GAAGAGCGTG 2220CGGGAGGCGG CCGAGCGCAT GGCCTTCAAC ATGCCCGTCC AGGGCACCGC CGCCGACCTC 2280ATGAAGCTGG CTATGGTGAA GCTCTTCCCC AGGCTGGAGG AAATGGGGGC CAGGATGCTC 2340CTTCAGGTCC ACGACGAGCT GGTCCTCGAG GCCCCAAAAG AGAGGGCGGA GGCCGTGGCC 2400CGGCTGGCCA AGGAGGTCAT GGAGGGGGTG TATCCCCTGG CCGTGCCCCT GGAGGTGGAG 2460GTGGGGATAG GGGAGGACTG GCTCTCCGCC AAGGAGTGAT ACCACC 2506 2496 base pairsnucleic acid double linear DNA (genomic) 2 ATGGCGATGC TTCCCCTCTTTGAGCCCAAA GGCCGCGTGC TCCTGGTGGA CGGCCACCAC 60 CTGGCCTACC GCACCTTCTTTGCCCTCAAG GGCCTCACCA CCAGCCGCGG CGAACCCGTT 120 CAGGCGGTCT ACGGCTTCGCCAAAAGCCTC CTCAAGGCCC TGAAGGAGGA CGGGGACGTG 180 GTGGTGGTGG TCTTTGACGCCAAGGCCCCC TCCTTCCGCC ACGAGGCCTA CGAGGCCTAC 240 AAGGCGGGCC GGGCCCCCACCCCGGAGGAC TTTCCCCGGC AGCTGGCCCT CATCAAGGAG 300 TTGGTGGACC TCCTAGGCCTTGTGCGGCTG GAGGTTCCCG GCTTTGAGGC GGACGACGTG 360 CTGGCCACCC TGGCCAAGCGGGCGGAAAAG GAGGGGTACG AGGTGCGCAT CCTCACTGCC 420 GACCGCGACC TCTACCAGCTCCTTTCGGAG CGCATCGCCA TCCTCCACCC TGAGGGGTAC 480 CTGATCACCC CGGCGTGGCTTTACGAGAAG TACGGCCTGC GCCCGGAGCA GTGGGTGGAC 540 TACCGGGCCC TGGCGGGGGACCCCTCGGAT AACATCCCCG GGGTGAAGGG CATCGGGGAG 600 AAGACCGCCC AGAGGCTCATCCGCGAGTGG GGGAGCCTGG AAAACCTCTT CCAGCACCTG 660 GACCAGGTGA AGCCCTCCTTGCGGGAGAAG CTCCAGGCGG GCATGGAGGC CCTGGCCCTT 720 TCCCGGAAGC TTTCCCAGGTGCACACTGAC CTGCCCCTGG AGGTGGACTT CGGGAGGCGC 780 CGCACACCCA ACCTGGAGGGTCTGCGGGCT TTTTTGGAGC GGTTGGAGTT TGGAAGCCTC 840 CTCCACGAGT TCGGCCTCCTGGAGGGGCCG AAGGCGGCAG AGGAGGCCCC CTGGCCCCCT 900 CCGGAAGGGG CTTTTTTGGGCTTTTCCTTT TCCCGTCCCG AGCCCATGTG GGCCGAGCTT 960 CTGGCCCTGG CTGGGGCGTGGGAGGGGCGC CTCCATCGGG CACAAGACCC CCTTAGGGGC 1020 CTGAGGGACC TTAAGGGGGTGCGGGGAATC CTGGCCAAGG ACCTGGCGGT TTTGGCCCTG 1080 CGGGAGGGCC TGGACCTCTTCCCAGAGGAC GACCCCATGC TCCTGGCCTA CCTTCTGGAC 1140 CCCTCCAACA CCACCCCTGAGGGGGTGGCC CGGCGTTACG GGGGGGAGTG GACGGAGGAT 1200 GCGGGGGAGA GGGCCCTCCTGGCCGAGCGC CTCTTCCAGA CCCTAAAGGA GCGCCTTAAG 1260 GGAGAAGAAC GCCTGCTTTGGCTTTACGAG GAGGTGGAGA AGCCGCTTTC CCGGGTGTTG 1320 GCCCGGATGG AGGCCACGGGGGTCCGGCTG GACGTGGCCT ACCTCCAGGC CCTCTCCCTG 1380 GAGGTGGAGG CGGAGGTGCGCCAGCTGGAG GAGGAGGTCT TCCGCCTGGC CGGCCACCCC 1440 TTCAACCTCA ACTCCCGCGACCAGCTGGAG CGGGTGCTCT TTGACGAGCT GGGCCTGCCT 1500 GCCATCGGCA AGACGGAGAAGACGGGGAAA CGCTCCACCA GCGCTGCCGT GCTGGAGGCC 1560 CTGCGAGAGG CCCACCCCATCGTGGACCGC ATCCTGCAGT ACCGGGAGCT CACCAAGCTC 1620 AAGAACACCT ACATAGACCCCCTGCCCGCC CTGGTCCACC CCAAGACCGG CCGGCTCCAC 1680 ACCCGCTTCA ACCAGACGGCCACCGCCACG GGCAGGCTTT CCAGCTCCGA CCCCAACCTG 1740 CAGAACATCC CCGTGCGCACCCCTCTGGGC CAGCGCATCC GCCGAGCCTT CGTGGCCGAG 1800 GAGGGCTGGG TGCTGGTGGTCTTGGACTAC AGCCAGATTG AGCTTCGGGT CCTGGCCCAC 1860 CTCTCCGGGG ACGAGAACCTGATCCGGGTC TTTCAGGAGG GGAGGGACAT CCACACCCAG 1920 ACCGCCAGCT GGATGTTCGGCGTTTCCCCC GAAGGGGTAG ACCCTCTGAT GCGCCGGGCG 1980 GCCAAGACCA TCAACTTCGGGGTGCTCTAC GGCATGTCCG CCCACCGCCT CTCCGGGGAG 2040 CTTTCCATCC CCTACGAGGAGGCGGTGGCC TTCATTGAGC GCTACTTCCA GAGCTACCCC 2100 AAGGTGCGGG CCTGGATTGAGGGGACCCTC GAGGAGGGCC GCCGGCGGGG GTATGTGGAG 2160 ACCCTCTTCG GCCGCCGGCGCTATGTGCCC GACCTCAACG CCCGGGTGAA GAGCGTGCGC 2220 GAGGCGGCGG AGCGCATGGCCTTCAACATG CCGGTCCAGG GCACCGCCGC CGACCTCATG 2280 AAGCTGGCCA TGGTGCGGCTTTTCCCCCGG CTTCAGGAAC TGGGGGCGAG GATGCTTTTG 2340 CAGGTGCACG ACGAGCTGGTCCTCGAGGCC CCCAAGGACC GGGCGGAGAG GGTAGCCGCT 2400 TTGGCCAAGG AGGTCATGGAGGGGGTCTGG CCCCTGCAGG TGCCCCTGGA GGTGGAGGTG 2460 GGCCTGGGGG AGGACTGGCTCTCCGCCAAG GAGTAG 2496 2504 base pairs nucleic acid double linear DNA(genomic) 3 ATGGAGGCGA TGCTTCCGCT CTTTGAACCC AAAGGCCGGG TCCTCCTGGTGGACGGCCAC 60 CACCTGGCCT ACCGCACCTT CTTCGCCCTG AAGGGCCTCA CCACGAGCCGGGGCGAACCG 120 GTGCAGGCGG TCTACGGCTT CGCCAAGAGC CTCCTCAAGG CCCTGAAGGAGGACGGGTAC 180 AAGGCCGTCT TCGTGGTCTT TGACGCCAAG GCCCCCTCCT TCCGCCACGAGGCCTACGAG 240 GCCTACAAGG CGGGGAGGGC CCCGACCCCC GAGGACTTCC CCCGGCAGCTCGCCCTCATC 300 AAGGAGCTGG TGGACCTCCT GGGGTTTACC CGCCTCGAGG TCCCCGGCTACGAGGCGGAC 360 GACGTTCTCG CCACCCTGGC CAAGAAGGCG GAAAAGGAGG GGTACGAGGTGCGCATCCTC 420 ACCGCCGACC GCGACCTCTA CCAACTCGTC TCCGACCGCG TCGCCGTCCTCCACCCCGAG 480 GGCCACCTCA TCACCCCGGA GTGGCTTTGG GAGAAGTACG GCCTCAGGCCGGAGCAGTGG 540 GTGGACTTCC GCGCCCTCGT GGGGGACCCC TCCGACAACC TCCCCGGGGTCAAGGGCATC 600 GGGGAGAAGA CCGCCCTCAA GCTCCTCAAG GAGTGGGGAA GCCTGGAAAACCTCCTCAAG 660 AACCTGGACC GGGTAAAGCC AGAAAACGTC CGGGAGAAGA TCAAGGCCCACCTGGAAGAC 720 CTCAGGCTCT CCTTGGAGCT CTCCCGGGTG CGCACCGACC TCCCCCTGGAGGTGGACCTC 780 GCCCAGGGGC GGGAGCCCGA CCGGGAGGGG CTTAGGGCCT TCCTGGAGAGGCTGGAGTTC 840 GGCAGCCTCC TCCACGAGTT CGGCCTCCTG GAGGCCCCCG CCCCCCTGGAGGAGGCCCCC 900 TGGCCCCCGC CGGAAGGGGC CTTCGTGGGC TTCGTCCTCT CCCGCCCCGAGCCCATGTGG 960 GCGGAGCTTA AAGCCCTGGC CGCCTGCAGG GACGGCCGGG TGCACCGGGCAGCAGACCCC 1020 TTGGCGGGGC TAAAGGACCT CAAGGAGGTC CGGGGCCTCC TCGCCAAGGACCTCGCCGTC 1080 TTGGCCTCGA GGGAGGGGCT AGACCTCGTG CCCGGGGACG ACCCCATGCTCCTCGCCTAC 1140 CTCCTGGACC CCTCCAACAC CACCCCCGAG GGGGTGGCGC GGCGCTACGGGGGGGAGTGG 1200 ACGGAGGACG CCGCCCACCG GGCCCTCCTC TCGGAGAGGC TCCATCGGAACCTCCTTAAG 1260 CGCCTCGAGG GGGAGGAGAA GCTCCTTTGG CTCTACCACG AGGTGGAAAAGCCCCTCTCC 1320 CGGGTCCTGG CCCACATGGA GGCCACCGGG GTACGGCTGG ACGTGGCCTACCTTCAGGCC 1380 CTTTCCCTGG AGCTTGCGGA GGAGATCCGC CGCCTCGAGG AGGAGGTCTTCCGCTTGGCG 1440 GGCCACCCCT TCAACCTCAA CTCCCGGGAC CAGCTGGAAA GGGTGCTCTTTGACGAGCTT 1500 AGGCTTCCCG CCTTGGGGAA GACGCAAAAG ACAGGCAAGC GCTCCACCAGCGCCGCGGTG 1560 CTGGAGGCCC TACGGGAGGC CCACCCCATC GTGGAGAAGA TCCTCCAGCACCGGGAGCTC 1620 ACCAAGCTCA AGAACACCTA CGTGGACCCC CTCCCAAGCC TCGTCCACCCGAGGACGGGC 1680 CGCCTCCACA CCCGCTTCAA CCAGACGGCC ACGGCCACGG GGAGGCTTAGTAGCTCCGAC 1740 CCCAACCTGC AGAACATCCC CGTCCGCACC CCCTTGGGCC AGAGGATCCGCCGGGCCTTC 1800 GTGGCCGAGG CGGGTTGGGC GTTGGTGGCC CTGGACTATA GCCAGATAGAGCTCCGCGTC 1860 CTCGCCCACC TCTCCGGGGA CGAAAACCTG ATCAGGGTCT TCCAGGAGGGGAAGGACATC 1920 CACACCCAGA CCGCAAGCTG GATGTTCGGC GTCCCCCCGG AGGCCGTGGACCCCCTGATG 1980 CGCCGGGCGG CCAAGACGGT GAACTTCGGC GTCCTCTACG GCATGTCCGCCCATAGGCTC 2040 TCCCAGGAGC TTGCCATCCC CTACGAGGAG GCGGTGGCCT TTATAGAGGCTACTTCCAAA 2100 GCTTCCCCAA GGTGCGGGCC TGGATAGAAA AGACCCTGGA GGAGGGGAGGAAGCGGGGCT 2160 ACGTGGAAAC CCTCTTCGGA AGAAGGCGCT ACGTGCCCGA CCTCAACGCCCGGGTGAAGA 2220 GCGTCAGGGA GGCCGCGGAG CGCATGGCCT TCAACATGCC CGTCCAGGGCACCGCCGCCG 2280 ACCTCATGAA GCTCGCCATG GTGAAGCTCT TCCCCCGCCT CCGGGAGATGGGGGCCCGCA 2340 TGCTCCTCCA GGTCCACGAC GAGCTCCTCC TGGAGGCCCC CCAAGCGCGGGCCGAGGAGG 2400 TGGCGGCTTT GGCCAAGGAG GCCATGGAGA AGGCCTATCC CCTCGCCGTGCCCCTGGAGG 2460 TGGAGGTGGG GATGGGGGAG GACTGGCTTT CCGCCAAGGG TTAG 2504832 amino acids amino acid single linear protein 4 Met Arg Gly Met LeuPro Leu Phe Glu Pro Lys Gly Arg Val Leu Leu 1 5 10 15 Val Asp Gly HisHis Leu Ala Tyr Arg Thr Phe His Ala Leu Lys Gly 20 25 30 Leu Thr Thr SerArg Gly Glu Pro Val Gln Ala Val Tyr Gly Phe Ala 35 40 45 Lys Ser Leu LeuLys Ala Leu Lys Glu Asp Gly Asp Ala Val Ile Val 50 55 60 Val Phe Asp AlaLys Ala Pro Ser Phe Arg His Glu Ala Tyr Gly Gly 65 70 75 80 Tyr Lys AlaGly Arg Ala Pro Thr Pro Glu Asp Phe Pro Arg Gln Leu 85 90 95 Ala Leu IleLys Glu Leu Val Asp Leu Leu Gly Leu Ala Arg Leu Glu 100 105 110 Val ProGly Tyr Glu Ala Asp Asp Val Leu Ala Ser Leu Ala Lys Lys 115 120 125 AlaGlu Lys Glu Gly Tyr Glu Val Arg Ile Leu Thr Ala Asp Lys Asp 130 135 140Leu Tyr Gln Leu Leu Ser Asp Arg Ile His Val Leu His Pro Glu Gly 145 150155 160 Tyr Leu Ile Thr Pro Ala Trp Leu Trp Glu Lys Tyr Gly Leu Arg Pro165 170 175 Asp Gln Trp Ala Asp Tyr Arg Ala Leu Thr Gly Asp Glu Ser AspAsn 180 185 190 Leu Pro Gly Val Lys Gly Ile Gly Glu Lys Thr Ala Arg LysLeu Leu 195 200 205 Glu Glu Trp Gly Ser Leu Glu Ala Leu Leu Lys Asn LeuAsp Arg Leu 210 215 220 Lys Pro Ala Ile Arg Glu Lys Ile Leu Ala His MetAsp Asp Leu Lys 225 230 235 240 Leu Ser Trp Asp Leu Ala Lys Val Arg ThrAsp Leu Pro Leu Glu Val 245 250 255 Asp Phe Ala Lys Arg Arg Glu Pro AspArg Glu Arg Leu Arg Ala Phe 260 265 270 Leu Glu Arg Leu Glu Phe Gly SerLeu Leu His Glu Phe Gly Leu Leu 275 280 285 Glu Ser Pro Lys Ala Leu GluGlu Ala Pro Trp Pro Pro Pro Glu Gly 290 295 300 Ala Phe Val Gly Phe ValLeu Ser Arg Lys Glu Pro Met Trp Ala Asp 305 310 315 320 Leu Leu Ala LeuAla Ala Ala Arg Gly Gly Arg Val His Arg Ala Pro 325 330 335 Glu Pro TyrLys Ala Leu Arg Asp Leu Lys Glu Ala Arg Gly Leu Leu 340 345 350 Ala LysAsp Leu Ser Val Leu Ala Leu Arg Glu Gly Leu Gly Leu Pro 355 360 365 ProGly Asp Asp Pro Met Leu Leu Ala Tyr Leu Leu Asp Pro Ser Asn 370 375 380Thr Thr Pro Glu Gly Val Ala Arg Arg Tyr Gly Gly Glu Trp Thr Glu 385 390395 400 Glu Ala Gly Glu Arg Ala Ala Leu Ser Glu Arg Leu Phe Ala Asn Leu405 410 415 Trp Gly Arg Leu Glu Gly Glu Glu Arg Leu Leu Trp Leu Tyr ArgGlu 420 425 430 Val Glu Arg Pro Leu Ser Ala Val Leu Ala His Met Glu AlaThr Gly 435 440 445 Val Arg Leu Asp Val Ala Tyr Leu Arg Ala Leu Ser LeuGlu Val Ala 450 455 460 Glu Glu Ile Ala Arg Leu Glu Ala Glu Val Phe ArgLeu Ala Gly His 465 470 475 480 Pro Phe Asn Leu Asn Ser Arg Asp Gln LeuGlu Arg Val Leu Phe Asp 485 490 495 Glu Leu Gly Leu Pro Ala Ile Gly LysThr Glu Lys Thr Gly Lys Arg 500 505 510 Ser Thr Ser Ala Ala Val Leu GluAla Leu Arg Glu Ala His Pro Ile 515 520 525 Val Glu Lys Ile Leu Gln TyrArg Glu Leu Thr Lys Leu Lys Ser Thr 530 535 540 Tyr Ile Asp Pro Leu ProAsp Leu Ile His Pro Arg Thr Gly Arg Leu 545 550 555 560 His Thr Arg PheAsn Gln Thr Ala Thr Ala Thr Gly Arg Leu Ser Ser 565 570 575 Ser Asp ProAsn Leu Gln Asn Ile Pro Val Arg Thr Pro Leu Gly Gln 580 585 590 Arg IleArg Arg Ala Phe Ile Ala Glu Glu Gly Trp Leu Leu Val Ala 595 600 605 LeuAsp Tyr Ser Gln Ile Glu Leu Arg Val Leu Ala His Leu Ser Gly 610 615 620Asp Glu Asn Leu Ile Arg Val Phe Gln Glu Gly Arg Asp Ile His Thr 625 630635 640 Glu Thr Ala Ser Trp Met Phe Gly Val Pro Arg Glu Ala Val Asp Pro645 650 655 Leu Met Arg Arg Ala Ala Lys Thr Ile Asn Phe Gly Val Leu TyrGly 660 665 670 Met Ser Ala His Arg Leu Ser Gln Glu Leu Ala Ile Pro TyrGlu Glu 675 680 685 Ala Gln Ala Phe Ile Glu Arg Tyr Phe Gln Ser Phe ProLys Val Arg 690 695 700 Ala Trp Ile Glu Lys Thr Leu Glu Glu Gly Arg ArgArg Gly Tyr Val 705 710 715 720 Glu Thr Leu Phe Gly Arg Arg Arg Tyr ValPro Asp Leu Glu Ala Arg 725 730 735 Val Lys Ser Val Arg Glu Ala Ala GluArg Met Ala Phe Asn Met Pro 740 745 750 Val Gln Gly Thr Ala Ala Asp LeuMet Lys Leu Ala Met Val Lys Leu 755 760 765 Phe Pro Arg Leu Glu Glu MetGly Ala Arg Met Leu Leu Gln Val His 770 775 780 Asp Glu Leu Val Leu GluAla Pro Lys Glu Arg Ala Glu Ala Val Ala 785 790 795 800 Arg Leu Ala LysGlu Val Met Glu Gly Val Tyr Pro Leu Ala Val Pro 805 810 815 Leu Glu ValGlu Val Gly Ile Gly Glu Asp Trp Leu Ser Ala Lys Glu 820 825 830 831amino acids amino acid single linear protein 5 Met Ala Met Leu Pro LeuPhe Glu Pro Lys Gly Arg Val Leu Leu Val 1 5 10 15 Asp Gly His His LeuAla Tyr Arg Thr Phe Phe Ala Leu Lys Gly Leu 20 25 30 Thr Thr Ser Arg GlyGlu Pro Val Gln Ala Val Tyr Gly Phe Ala Lys 35 40 45 Ser Leu Leu Lys AlaLeu Lys Glu Asp Gly Asp Val Val Val Val Val 50 55 60 Phe Asp Ala Lys AlaPro Ser Phe Arg His Glu Ala Tyr Glu Ala Tyr 65 70 75 80 Lys Ala Gly ArgAla Pro Thr Pro Glu Asp Phe Pro Arg Gln Leu Ala 85 90 95 Leu Ile Lys GluLeu Val Asp Leu Leu Gly Leu Val Arg Leu Glu Val 100 105 110 Pro Gly PheGlu Ala Asp Asp Val Leu Ala Thr Leu Ala Lys Arg Ala 115 120 125 Glu LysGlu Gly Tyr Glu Val Arg Ile Leu Thr Ala Asp Arg Asp Leu 130 135 140 TyrGln Leu Leu Ser Glu Arg Ile Ala Ile Leu His Pro Glu Gly Tyr 145 150 155160 Leu Ile Thr Pro Ala Trp Leu Tyr Glu Lys Tyr Gly Leu Arg Pro Glu 165170 175 Gln Trp Val Asp Tyr Arg Ala Leu Ala Gly Asp Pro Ser Asp Asn Ile180 185 190 Pro Gly Val Lys Gly Ile Gly Glu Lys Thr Ala Gln Arg Leu IleArg 195 200 205 Glu Trp Gly Ser Leu Glu Asn Leu Phe Gln His Leu Asp GlnVal Lys 210 215 220 Pro Ser Leu Arg Glu Lys Leu Gln Ala Gly Met Glu AlaLeu Ala Leu 225 230 235 240 Ser Arg Lys Leu Ser Gln Val His Thr Asp LeuPro Leu Glu Val Asp 245 250 255 Phe Gly Arg Arg Arg Thr Pro Asn Leu GluGly Leu Arg Ala Phe Leu 260 265 270 Glu Arg Leu Glu Phe Gly Ser Leu LeuHis Glu Phe Gly Leu Leu Glu 275 280 285 Gly Pro Lys Ala Ala Glu Glu AlaPro Trp Pro Pro Pro Glu Gly Ala 290 295 300 Phe Leu Gly Phe Ser Phe SerArg Pro Glu Pro Met Trp Ala Glu Leu 305 310 315 320 Leu Ala Leu Ala GlyAla Trp Glu Gly Arg Leu His Arg Ala Gln Asp 325 330 335 Pro Leu Arg GlyLeu Arg Asp Leu Lys Gly Val Arg Gly Ile Leu Ala 340 345 350 Lys Asp LeuAla Val Leu Ala Leu Arg Glu Gly Leu Asp Leu Phe Pro 355 360 365 Glu AspAsp Pro Met Leu Leu Ala Tyr Leu Leu Asp Pro Ser Asn Thr 370 375 380 ThrPro Glu Gly Val Ala Arg Arg Tyr Gly Gly Glu Trp Thr Glu Asp 385 390 395400 Ala Gly Glu Arg Ala Leu Leu Ala Glu Arg Leu Phe Gln Thr Leu Lys 405410 415 Glu Arg Leu Lys Gly Glu Glu Arg Leu Leu Trp Leu Tyr Glu Glu Val420 425 430 Glu Lys Pro Leu Ser Arg Val Leu Ala Arg Met Glu Ala Thr GlyVal 435 440 445 Arg Leu Asp Val Ala Tyr Leu Gln Ala Leu Ser Leu Glu ValGlu Ala 450 455 460 Glu Val Arg Gln Leu Glu Glu Glu Val Phe Arg Leu AlaGly His Pro 465 470 475 480 Phe Asn Leu Asn Ser Arg Asp Gln Leu Glu ArgVal Leu Phe Asp Glu 485 490 495 Leu Gly Leu Pro Ala Ile Gly Lys Thr GluLys Thr Gly Lys Arg Ser 500 505 510 Thr Ser Ala Ala Val Leu Glu Ala LeuArg Glu Ala His Pro Ile Val 515 520 525 Asp Arg Ile Leu Gln Tyr Arg GluLeu Thr Lys Leu Lys Asn Thr Tyr 530 535 540 Ile Asp Pro Leu Pro Ala LeuVal His Pro Lys Thr Gly Arg Leu His 545 550 555 560 Thr Arg Phe Asn GlnThr Ala Thr Ala Thr Gly Arg Leu Ser Ser Ser 565 570 575 Asp Pro Asn LeuGln Asn Ile Pro Val Arg Thr Pro Leu Gly Gln Arg 580 585 590 Ile Arg ArgAla Phe Val Ala Glu Glu Gly Trp Val Leu Val Val Leu 595 600 605 Asp TyrSer Gln Ile Glu Leu Arg Val Leu Ala His Leu Ser Gly Asp 610 615 620 GluAsn Leu Ile Arg Val Phe Gln Glu Gly Arg Asp Ile His Thr Gln 625 630 635640 Thr Ala Ser Trp Met Phe Gly Val Ser Pro Glu Gly Val Asp Pro Leu 645650 655 Met Arg Arg Ala Ala Lys Thr Ile Asn Phe Gly Val Leu Tyr Gly Met660 665 670 Ser Ala His Arg Leu Ser Gly Glu Leu Ser Ile Pro Tyr Glu GluAla 675 680 685 Val Ala Phe Ile Glu Arg Tyr Phe Gln Ser Tyr Pro Lys ValArg Ala 690 695 700 Trp Ile Glu Gly Thr Leu Glu Glu Gly Arg Arg Arg GlyTyr Val Glu 705 710 715 720 Thr Leu Phe Gly Arg Arg Arg Tyr Val Pro AspLeu Asn Ala Arg Val 725 730 735 Lys Ser Val Arg Glu Ala Ala Glu Arg MetAla Phe Asn Met Pro Val 740 745 750 Gln Gly Thr Ala Ala Asp Leu Met LysLeu Ala Met Val Arg Leu Phe 755 760 765 Pro Arg Leu Gln Glu Leu Gly AlaArg Met Leu Leu Gln Val His Asp 770 775 780 Glu Leu Val Leu Glu Ala ProLys Asp Arg Ala Glu Arg Val Ala Ala 785 790 795 800 Leu Ala Lys Glu ValMet Glu Gly Val Trp Pro Leu Gln Val Pro Leu 805 810 815 Glu Val Glu ValGly Leu Gly Glu Asp Trp Leu Ser Ala Lys Glu 820 825 830 834 amino acidsamino acid single linear protein 6 Met Glu Ala Met Leu Pro Leu Phe GluPro Lys Gly Arg Val Leu Leu 1 5 10 15 Val Asp Gly His His Leu Ala TyrArg Thr Phe Phe Ala Leu Lys Gly 20 25 30 Leu Thr Thr Ser Arg Gly Glu ProVal Gln Ala Val Tyr Gly Phe Ala 35 40 45 Lys Ser Leu Leu Lys Ala Leu LysGlu Asp Gly Tyr Lys Ala Val Phe 50 55 60 Val Val Phe Asp Ala Lys Ala ProSer Phe Arg His Glu Ala Tyr Glu 65 70 75 80 Ala Tyr Lys Ala Gly Arg AlaPro Thr Pro Glu Asp Phe Pro Arg Gln 85 90 95 Leu Ala Leu Ile Lys Glu LeuVal Asp Leu Leu Gly Phe Thr Arg Leu 100 105 110 Glu Val Pro Gly Tyr GluAla Asp Asp Val Leu Ala Thr Leu Ala Lys 115 120 125 Lys Ala Glu Lys GluGly Tyr Glu Val Arg Ile Leu Thr Ala Asp Arg 130 135 140 Asp Leu Tyr GlnLeu Val Ser Asp Arg Val Ala Val Leu His Pro Glu 145 150 155 160 Gly HisLeu Ile Thr Pro Glu Trp Leu Trp Glu Lys Tyr Gly Leu Arg 165 170 175 ProGlu Gln Trp Val Asp Phe Arg Ala Leu Val Gly Asp Pro Ser Asp 180 185 190Asn Leu Pro Gly Val Lys Gly Ile Gly Glu Lys Thr Ala Leu Lys Leu 195 200205 Leu Lys Glu Trp Gly Ser Leu Glu Asn Leu Leu Lys Asn Leu Asp Arg 210215 220 Val Lys Pro Glu Asn Val Arg Glu Lys Ile Lys Ala His Leu Glu Asp225 230 235 240 Leu Arg Leu Ser Leu Glu Leu Ser Arg Val Arg Thr Asp LeuPro Leu 245 250 255 Glu Val Asp Leu Ala Gln Gly Arg Glu Pro Asp Arg GluGly Leu Arg 260 265 270 Ala Phe Leu Glu Arg Leu Glu Phe Gly Ser Leu LeuHis Glu Phe Gly 275 280 285 Leu Leu Glu Ala Pro Ala Pro Leu Glu Glu AlaPro Trp Pro Pro Pro 290 295 300 Glu Gly Ala Phe Val Gly Phe Val Leu SerArg Pro Glu Pro Met Trp 305 310 315 320 Ala Glu Leu Lys Ala Leu Ala AlaCys Arg Asp Gly Arg Val His Arg 325 330 335 Ala Ala Asp Pro Leu Ala GlyLeu Lys Asp Leu Lys Glu Val Arg Gly 340 345 350 Leu Leu Ala Lys Asp LeuAla Val Leu Ala Ser Arg Glu Gly Leu Asp 355 360 365 Leu Val Pro Gly AspAsp Pro Met Leu Leu Ala Tyr Leu Leu Asp Pro 370 375 380 Ser Asn Thr ThrPro Glu Gly Val Ala Arg Arg Tyr Gly Gly Glu Trp 385 390 395 400 Thr GluAsp Ala Ala His Arg Ala Leu Leu Ser Glu Arg Leu His Arg 405 410 415 AsnLeu Leu Lys Arg Leu Glu Gly Glu Glu Lys Leu Leu Trp Leu Tyr 420 425 430His Glu Val Glu Lys Pro Leu Ser Arg Val Leu Ala His Met Glu Ala 435 440445 Thr Gly Val Arg Leu Asp Val Ala Tyr Leu Gln Ala Leu Ser Leu Glu 450455 460 Leu Ala Glu Glu Ile Arg Arg Leu Glu Glu Glu Val Phe Arg Leu Ala465 470 475 480 Gly His Pro Phe Asn Leu Asn Ser Arg Asp Gln Leu Glu ArgVal Leu 485 490 495 Phe Asp Glu Leu Arg Leu Pro Ala Leu Gly Lys Thr GlnLys Thr Gly 500 505 510 Lys Arg Ser Thr Ser Ala Ala Val Leu Glu Ala LeuArg Glu Ala His 515 520 525 Pro Ile Val Glu Lys Ile Leu Gln His Arg GluLeu Thr Lys Leu Lys 530 535 540 Asn Thr Tyr Val Asp Pro Leu Pro Ser LeuVal His Pro Arg Thr Gly 545 550 555 560 Arg Leu His Thr Arg Phe Asn GlnThr Ala Thr Ala Thr Gly Arg Leu 565 570 575 Ser Ser Ser Asp Pro Asn LeuGln Asn Ile Pro Val Arg Thr Pro Leu 580 585 590 Gly Gln Arg Ile Arg ArgAla Phe Val Ala Glu Ala Gly Trp Ala Leu 595 600 605 Val Ala Leu Asp TyrSer Gln Ile Glu Leu Arg Val Leu Ala His Leu 610 615 620 Ser Gly Asp GluAsn Leu Ile Arg Val Phe Gln Glu Gly Lys Asp Ile 625 630 635 640 His ThrGln Thr Ala Ser Trp Met Phe Gly Val Pro Pro Glu Ala Val 645 650 655 AspPro Leu Met Arg Arg Ala Ala Lys Thr Val Asn Phe Gly Val Leu 660 665 670Tyr Gly Met Ser Ala His Arg Leu Ser Gln Glu Leu Ala Ile Pro Tyr 675 680685 Glu Glu Ala Val Ala Phe Ile Glu Arg Tyr Phe Gln Ser Phe Pro Lys 690695 700 Val Arg Ala Trp Ile Glu Lys Thr Leu Glu Glu Gly Arg Lys Arg Gly705 710 715 720 Tyr Val Glu Thr Leu Phe Gly Arg Arg Arg Tyr Val Pro AspLeu Asn 725 730 735 Ala Arg Val Lys Ser Val Arg Glu Ala Ala Glu Arg MetAla Phe Asn 740 745 750 Met Pro Val Gln Gly Thr Ala Ala Asp Leu Met LysLeu Ala Met Val 755 760 765 Lys Leu Phe Pro Arg Leu Arg Glu Met Gly AlaArg Met Leu Leu Gln 770 775 780 Val His Asp Glu Leu Leu Leu Glu Ala ProGln Ala Arg Ala Glu Glu 785 790 795 800 Val Ala Ala Leu Ala Lys Glu AlaMet Glu Lys Ala Tyr Pro Leu Ala 805 810 815 Val Pro Leu Glu Val Glu ValGly Met Gly Glu Asp Trp Leu Ser Ala 820 825 830 Lys Gly 2502 base pairsnucleic acid single linear DNA (genomic) 7 ATGNNGGCGA TGCTTCCCCTCTTTGAGCCC AAAGGCCGGG TCCTCCTGGT GGACGGCCAC 60 CACCTGGCCT ACCGCACCTTCTTCGCCCTG AAGGGCCTCA CCACCAGCCG GGGCGAACCG 120 GTGCAGGCGG TCTACGGCTTCGCCAAGAGC CTCCTCAAGG CCCTGAAGGA GGACGGGGAC 180 NNGGCGGTGN TCGTGGTCTTTGACGCCAAG GCCCCCTCCT TCCGCCACGA GGCCTACGAG 240 GCCTACAAGG CGGGCCGGGCCCCCACCCCG GAGGACTTTC CCCGGCAGCT CGCCCTCATC 300 AAGGAGCTGG TGGACCTCCTGGGGCTTGCG CGCCTCGAGG TCCCCGGCTA CGAGGCGGAC 360 GACGTNCTGG CCACCCTGGCCAAGAAGGCG GAAAAGGAGG GGTACGAGGT GCGCATCCTC 420 ACCGCCGACC GCGACCTCTACCAGCTCCTT TCCGACCGCA TCGCCGTCCT CCACCCCGAG 480 GGGTACCTCA TCACCCCGGCGTGGCTTTGG GAGAAGTACG GCCTGAGGCC GGAGCAGTGG 540 GTGGACTACC GGGCCCTGGCGGGGGACCCC TCCGACAACC TCCCCGGGGT CAAGGGCATC 600 GGGGAGAAGA CCGCCCNGAAGCTCCTCNAG GAGTGGGGGA GCCTGGAAAA CCTCCTCAAG 660 AACCTGGACC GGGTGAAGCCCGCCNTCCGG GAGAAGATCC AGGCCCACAT GGANGACCTG 720 ANGCTCTCCT GGGAGCTNTCCCAGGTGCGC ACCGACCTGC CCCTGGAGGT GGACTTCGCC 780 AAGNGGCGGG AGCCCGACCGGGAGGGGCTT AGGGCCTTTC TGGAGAGGCT GGAGTTTGGC 840 AGCCTCCTCC ACGAGTTCGGCCTCCTGGAG GGCCCCAAGG CCCTGGAGGA GGCCCCCTGG 900 CCCCCGCCGG AAGGGGCCTTCGTGGGCTTT GTCCTTTCCC GCCCCGAGCC CATGTGGGCC 960 GAGCTTCTGG CCCTGGCCGCCGCCAGGGAG GGCCGGGTCC ACCGGGCACC AGACCCCTTT 1020 ANGGGCCTNA GGGACCTNAAGGAGGTGCGG GGNCTCCTCG CCAAGGACCT GGCCGTTTTG 1080 GCCCTGAGGG AGGGCCTNGACCTCNTGCCC GGGGACGACC CCATGCTCCT CGCCTACCTC 1140 CTGGACCCCT CCAACACCACCCCCGAGGGG GTGGCCCGGC GCTACGGGGG GGAGTGGACG 1200 GAGGANGCGG GGGAGCGGGCCCTCCTNTCC GAGAGGCTCT TCCNGAACCT NNNGCAGCGC 1260 CTTGAGGGGG AGGAGAGGCTCCTTTGGCTT TACCAGGAGG TGGAGAAGCC CCTTTCCCGG 1320 GTCCTGGCCC ACATGGAGGCCACGGGGGTN CGGCTGGACG TGGCCTACCT CCAGGCCCTN 1380 TCCCTGGAGG TGGCGGAGGAGATCCGCCGC CTCGAGGAGG AGGTCTTCCG CCTGGCCGGC 1440 CACCCCTTCA ACCTCAACTCCCGGGACCAG CTGGAAAGGG TGCTCTTTGA CGAGCTNGGG 1500 CTTCCCGCCA TCGGCAAGACGGAGAAGACN GGCAAGCGCT CCACCAGCGC CGCCGTGCTG 1560 GAGGCCCTNC GNGAGGCCCACCCCATCGTG GAGAAGATCC TGCAGTACCG GGAGCTCACC 1620 AAGCTCAAGA ACACCTACATNGACCCCCTG CCNGNCCTCG TCCACCCCAG GACGGGCCGC 1680 CTCCACACCC GCTTCAACCAGACGGCCACG GCCACGGGCA GGCTTAGTAG CTCCGACCCC 1740 AACCTGCAGA ACATCCCCGTCCGCACCCCN CTGGGCCAGA GGATCCGCCG GGCCTTCGTG 1800 GCCGAGGAGG GNTGGGTGTTGGTGGCCCTG GACTATAGCC AGATAGAGCT CCGGGTCCTG 1860 GCCCACCTCT CCGGGGACGAGAACCTGATC CGGGTCTTCC AGGAGGGGAG GGACATCCAC 1920 ACCCAGACCG CCAGCTGGATGTTCGGCGTC CCCCCGGAGG CCGTGGACCC CCTGATGCGC 1980 CGGGCGGCCA AGACCATCAACTTCGGGGTC CTCTACGGCA TGTCCGCCCA CCGCCTCTCC 2040 CAGGAGCTTG CCATCCCCTACGAGGAGGCG GTGGCCTTCA TTGAGCGCTA CTTCCAGAGC 2100 TTCCCCAAGG TGCGGGCCTGGATTGAGAAG ACCCTGGAGG AGGGCAGGAG GCGGGGGTAC 2160 GTGGAGACCC TCTTCGGCCGCCGGCGCTAC GTGCCCGACC TCAACGCCCG GGTGAAGAGC 2220 GTGCGGGAGG CGGCGGAGCGCATGGCCTTC AACATGCCCG TCCAGGGCAC CGCCGCCGAC 2280 CTCATGAAGC TGGCCATGGTGAAGCTCTTC CCCCGGCTNC AGGAAATGGG GGCCAGGATG 2340 CTCCTNCAGG TCCACGACGAGCTGGTCCTC GAGGCCCCCA AAGAGCGGGC GGAGGNGGTG 2400 GCCGCTTTGG CCAAGGAGGTCATGGAGGGG GTCTATCCCC TGGCCGTGCC CCTGGAGGTG 2460 GAGGTGGGGA TGGGGGAGGACTGGCTCTCC GCCAAGGAGT AG 2502 833 amino acids amino acid single unknownpeptide 8 Met Xaa Ala Met Leu Pro Leu Phe Glu Pro Lys Gly Arg Val LeuLeu 1 5 10 15 Val Asp Gly His His Leu Ala Tyr Arg Thr Phe Phe Ala LeuLys Gly 20 25 30 Leu Thr Thr Ser Arg Gly Glu Pro Val Gln Ala Val Tyr GlyPhe Ala 35 40 45 Lys Ser Leu Leu Lys Ala Leu Lys Glu Asp Gly Asp Ala ValXaa Val 50 55 60 Val Phe Asp Ala Lys Ala Pro Ser Phe Arg His Glu Ala TyrGlu Ala 65 70 75 80 Tyr Lys Ala Gly Arg Ala Pro Thr Pro Glu Asp Phe ProArg Gln Leu 85 90 95 Ala Leu Ile Lys Glu Leu Val Asp Leu Leu Gly Leu XaaArg Leu Glu 100 105 110 Val Pro Gly Tyr Glu Ala Asp Asp Val Leu Ala ThrLeu Ala Lys Lys 115 120 125 Ala Glu Lys Glu Gly Tyr Glu Val Arg Ile LeuThr Ala Asp Arg Asp 130 135 140 Leu Tyr Gln Leu Leu Ser Asp Arg Ile AlaVal Leu His Pro Glu Gly 145 150 155 160 Tyr Leu Ile Thr Pro Ala Trp LeuTrp Glu Lys Tyr Gly Leu Arg Pro 165 170 175 Glu Gln Trp Val Asp Tyr ArgAla Leu Xaa Gly Asp Pro Ser Asp Asn 180 185 190 Leu Pro Gly Val Lys GlyIle Gly Glu Lys Thr Ala Xaa Lys Leu Leu 195 200 205 Xaa Glu Trp Gly SerLeu Glu Asn Leu Leu Lys Asn Leu Asp Arg Val 210 215 220 Lys Pro Xaa XaaArg Glu Lys Ile Xaa Ala His Met Glu Asp Leu Xaa 225 230 235 240 Leu SerXaa Xaa Leu Ser Xaa Val Arg Thr Asp Leu Pro Leu Glu Val 245 250 255 AspPhe Ala Xaa Arg Arg Glu Pro Asp Arg Glu Gly Leu Arg Ala Phe 260 265 270Leu Glu Arg Leu Glu Phe Gly Ser Leu Leu His Glu Phe Gly Leu Leu 275 280285 Glu Xaa Pro Lys Ala Leu Glu Glu Ala Pro Trp Pro Pro Pro Glu Gly 290295 300 Ala Phe Val Gly Phe Val Leu Ser Arg Pro Glu Pro Met Trp Ala Glu305 310 315 320 Leu Leu Ala Leu Ala Ala Ala Arg Xaa Gly Arg Val His ArgAla Xaa 325 330 335 Asp Pro Leu Xaa Gly Leu Arg Asp Leu Lys Glu Val ArgGly Leu Leu 340 345 350 Ala Lys Asp Leu Ala Val Leu Ala Leu Arg Glu GlyLeu Asp Leu Xaa 355 360 365 Pro Gly Asp Asp Pro Met Leu Leu Ala Tyr LeuLeu Asp Pro Ser Asn 370 375 380 Thr Thr Pro Glu Gly Val Ala Arg Arg TyrGly Gly Glu Trp Thr Glu 385 390 395 400 Asp Ala Gly Glu Arg Ala Leu LeuSer Glu Arg Leu Phe Xaa Asn Leu 405 410 415 Xaa Xaa Arg Leu Glu Gly GluGlu Arg Leu Leu Trp Leu Tyr Xaa Glu 420 425 430 Val Glu Lys Pro Leu SerArg Val Leu Ala His Met Glu Ala Thr Gly 435 440 445 Val Arg Leu Asp ValAla Tyr Leu Gln Ala Leu Ser Leu Glu Val Ala 450 455 460 Glu Glu Ile ArgArg Leu Glu Glu Glu Val Phe Arg Leu Ala Gly His 465 470 475 480 Pro PheAsn Leu Asn Ser Arg Asp Gln Leu Glu Arg Val Leu Phe Asp 485 490 495 GluLeu Gly Leu Pro Ala Ile Gly Lys Thr Glu Lys Thr Gly Lys Arg 500 505 510Ser Thr Ser Ala Ala Val Leu Glu Ala Leu Arg Glu Ala His Pro Ile 515 520525 Val Glu Lys Ile Leu Gln Tyr Arg Glu Leu Thr Lys Leu Lys Asn Thr 530535 540 Tyr Ile Asp Pro Leu Pro Xaa Leu Val His Pro Arg Thr Gly Arg Leu545 550 555 560 His Thr Arg Phe Asn Gln Thr Ala Thr Ala Thr Gly Arg LeuSer Ser 565 570 575 Ser Asp Pro Asn Leu Gln Asn Ile Pro Val Arg Thr ProLeu Gly Gln 580 585 590 Arg Ile Arg Arg Ala Phe Val Ala Glu Glu Gly TrpXaa Leu Val Ala 595 600 605 Leu Asp Tyr Ser Gln Ile Glu Leu Arg Val LeuAla His Leu Ser Gly 610 615 620 Asp Glu Asn Leu Ile Arg Val Phe Gln GluGly Arg Asp Ile His Thr 625 630 635 640 Gln Thr Ala Ser Trp Met Phe GlyVal Pro Pro Glu Ala Val Asp Pro 645 650 655 Leu Met Arg Arg Ala Ala LysThr Ile Asn Phe Gly Val Leu Tyr Gly 660 665 670 Met Ser Ala His Arg LeuSer Gln Glu Leu Ala Ile Pro Tyr Glu Glu 675 680 685 Ala Val Ala Phe IleGlu Arg Tyr Phe Gln Ser Phe Pro Lys Val Arg 690 695 700 Ala Trp Ile GluLys Thr Leu Glu Glu Gly Arg Arg Arg Gly Tyr Val 705 710 715 720 Glu ThrLeu Phe Gly Arg Arg Arg Tyr Val Pro Asp Leu Asn Ala Arg 725 730 735 ValLys Ser Val Arg Glu Ala Ala Glu Arg Met Ala Phe Asn Met Pro 740 745 750Val Gln Gly Thr Ala Ala Asp Leu Met Lys Leu Ala Met Val Lys Leu 755 760765 Phe Pro Arg Leu Xaa Glu Met Gly Ala Arg Met Leu Leu Gln Val His 770775 780 Asp Glu Leu Val Leu Glu Ala Pro Lys Xaa Arg Ala Glu Xaa Val Ala785 790 795 800 Ala Leu Ala Lys Glu Val Met Glu Gly Val Tyr Pro Leu AlaVal Pro 805 810 815 Leu Glu Val Glu Val Gly Xaa Gly Glu Asp Trp Leu SerAla Lys Glu 820 825 830 Xaa 1647 base pairs nucleic acid double linearDNA (genomic) 9 ATGAATTCGG GGATGCTGCC CCTCTTTGAG CCCAAGGGCC GGGTCCTCCTGGTGGACGGC 60 CACCACCTGG CCTACCGCAC CTTCCACGCC CTGAAGGGCC TCACCACCAGCCGGGGGGAG 120 CCGGTGCAGG CGGTCTACGG CTTCGCCAAG AGCCTCCTCA AGGCCCTCAAGGAGGACGGG 180 GACGCGGTGA TCGTGGTCTT TGACGCCAAG GCCCCCTCCT TCCGCCACGAGGCCTACGGG 240 GGGTACAAGG CGGGCCGGGC CCCCACGCCG GAGGACTTTC CCCGGCAACTCGCCCTCATC 300 AAGGAGCTGG TGGACCTCCT GGGGCTGGCG CGCCTCGAGG TCCCGGGCTACGAGGCGGAC 360 GACGTCCTGG CCAGCCTGGC CAAGAAGGCG GAAAAGGAGG GCTACGAGGTCCGCATCCTC 420 ACCGCCGACA AAGACCTTTA CCAGCTCCTT TCCGACCGCA TCCACGTCCTCCACCCCGAG 480 GGGTACCTCA TCACCCCGGC CTGGCTTTGG GAAAAGTACG GCCTGAGGCCCGACCAGTGG 540 GCCGACTACC GGGCCCTGAC CGGGGACGAG TCCGACAACC TTCCCGGGGTCAAGGGCATC 600 GGGGAGAAGA CGGCGAGGAA GCTTCTGGAG GAGTGGGGGA GCCTGGAAGCCCTCCTCAAG 660 AACCTGGACC GGCTGAAGCC CGCCATCCGG GAGAAGATCC TGGCCCACATGGACGATCTG 720 AAGCTCTCCT GGGACCTGGC CAAGGTGCGC ACCGACCTGC CCCTGGAGGTGGACTTCGCC 780 AAAAGGCGGG AGCCCGACCG GGAGAGGCTT AGGGCCTTTC TGGAGAGGCTTGAGTTTGGC 840 AGCCTCCTCC ACGAGTTCGG CCTTCTGGAA AGCCCCAAGG CCCTGGAGGAGGCCCCCTGG 900 CCCCCGCCGG AAGGGGCCTT CGTGGGCTTT GTGCTTTCCC GCAAGGAGCCCATGTGGGCC 960 GATCTTCTGG CCCTGGCCGC CGCCAGGGGG GGCCGGGTCC ACCGGGCCCCCGAGCCTTAT 1020 AAAGCCCTCA GGGACCTGAA GGAGGCGCGG GGGCTTCTCG CCAAAGACCTGAGCGTTCTG 1080 GCCCTGAGGG AAGGCCTTGG CCTCCCGCCC GGCGACGACC CCATGCTCCTCGCCTACCTC 1140 CTGGACCCTT CCAACACCAC CCCCGAGGGG GTGGCCCGGC GCTACGGCGGGGAGTGGACG 1200 GAGGAGGCGG GGGAGCGGGC CGCCCTTTCC GAGAGGCTCT TCGCCAACCTGTGGGGGAGG 1260 CTTGAGGGGG AGGAGAGGCT CCTTTGGCTT TACCGGGAGG TGGAGAGGCCCCTTTCCGCT 1320 GTCCTGGCCC ACATGGAGGC CACGGGGGTG CGCCTGGACG TGGCCTATCTCAGGGCCTTG 1380 TCCCTGGAGG TGGCCGGGGA GATCGCCCGC CTCGAGGCCG AGGTCTTCCGCCTGGCCGGC 1440 CACCCCTTCA ACCTCAACTC CCGGGACCAG CTGGAAAGGG TCCTCTTTGACGAGCTAGGG 1500 CTTCCCGCCA TCGGCAAGAC GGAGAAGACC GGCAAGCGCT CCACCAGCGCCGCCGTCCTG 1560 GAGGCCCTCC GCGAGGCCCA CCCCATCGTG GAGAAGATCC TGCAGGCATGCAAGCTTGGC 1620 ACTGGCCGTC GTTTTACAAC GTCGTGA 1647 2088 base pairsnucleic acid double linear DNA (genomic) 10 ATGAATTCGG GGATGCTGCCCCTCTTTGAG CCCAAGGGCC GGGTCCTCCT GGTGGACGGC 60 CACCACCTGG CCTACCGCACCTTCCACGCC CTGAAGGGCC TCACCACCAG CCGGGGGGAG 120 CCGGTGCAGG CGGTCTACGGCTTCGCCAAG AGCCTCCTCA AGGCCCTCAA GGAGGACGGG 180 GACGCGGTGA TCGTGGTCTTTGACGCCAAG GCCCCCTCCT TCCGCCACGA GGCCTACGGG 240 GGGTACAAGG CGGGCCGGGCCCCCACGCCG GAGGACTTTC CCCGGCAACT CGCCCTCATC 300 AAGGAGCTGG TGGACCTCCTGGGGCTGGCG CGCCTCGAGG TCCCGGGCTA CGAGGCGGAC 360 GACGTCCTGG CCAGCCTGGCCAAGAAGGCG GAAAAGGAGG GCTACGAGGT CCGCATCCTC 420 ACCGCCGACA AAGACCTTTACCAGCTCCTT TCCGACCGCA TCCACGTCCT CCACCCCGAG 480 GGGTACCTCA TCACCCCGGCCTGGCTTTGG GAAAAGTACG GCCTGAGGCC CGACCAGTGG 540 GCCGACTACC GGGCCCTGACCGGGGACGAG TCCGACAACC TTCCCGGGGT CAAGGGCATC 600 GGGGAGAAGA CGGCGAGGAAGCTTCTGGAG GAGTGGGGGA GCCTGGAAGC CCTCCTCAAG 660 AACCTGGACC GGCTGAAGCCCGCCATCCGG GAGAAGATCC TGGCCCACAT GGACGATCTG 720 AAGCTCTCCT GGGACCTGGCCAAGGTGCGC ACCGACCTGC CCCTGGAGGT GGACTTCGCC 780 AAAAGGCGGG AGCCCGACCGGGAGAGGCTT AGGGCCTTTC TGGAGAGGCT TGAGTTTGGC 840 AGCCTCCTCC ACGAGTTCGGCCTTCTGGAA AGCCCCAAGG CCCTGGAGGA GGCCCCCTGG 900 CCCCCGCCGG AAGGGGCCTTCGTGGGCTTT GTGCTTTCCC GCAAGGAGCC CATGTGGGCC 960 GATCTTCTGG CCCTGGCCGCCGCCAGGGGG GGCCGGGTCC ACCGGGCCCC CGAGCCTTAT 1020 AAAGCCCTCA GGGACCTGAAGGAGGCGCGG GGGCTTCTCG CCAAAGACCT GAGCGTTCTG 1080 GCCCTGAGGG AAGGCCTTGGCCTCCCGCCC GGCGACGACC CCATGCTCCT CGCCTACCTC 1140 CTGGACCCTT CCAACACCACCCCCGAGGGG GTGGCCCGGC GCTACGGCGG GGAGTGGACG 1200 GAGGAGGCGG GGGAGCGGGCCGCCCTTTCC GAGAGGCTCT TCGCCAACCT GTGGGGGAGG 1260 CTTGAGGGGG AGGAGAGGCTCCTTTGGCTT TACCGGGAGG TGGAGAGGCC CCTTTCCGCT 1320 GTCCTGGCCC ACATGGAGGCCACGGGGGTG CGCCTGGACG TGGCCTATCT CAGGGCCTTG 1380 TCCCTGGAGG TGGCCGGGGAGATCGCCCGC CTCGAGGCCG AGGTCTTCCG CCTGGCCGGC 1440 CACCCCTTCA ACCTCAACTCCCGGGACCAG CTGGAAAGGG TCCTCTTTGA CGAGCTAGGG 1500 CTTCCCGCCA TCGGCAAGACGGAGAAGACC GGCAAGCGCT CCACCAGCGC CGCCGTCCTG 1560 GAGGCCCTCC GCGAGGCCCACCCCATCGTG GAGAAGATCC TGCAGTACCG GGAGCTCACC 1620 AAGCTGAAGA GCACCTACATTGACCCCTTG CCGGACCTCA TCCACCCCAG GACGGGCCGC 1680 CTCCACACCC GCTTCAACCAGACGGCCACG GCCACGGGCA GGCTAAGTAG CTCCGATCCC 1740 AACCTCCAGA ACATCCCCGTCCGCACCCCG CTTGGGCAGA GGATCCGCCG GGCCTTCATC 1800 GCCGAGGAGG GGTGGCTATTGGTGGCCCTG GACTATAGCC AGATAGAGCT CAGGGTGCTG 1860 GCCCACCTCT CCGGCGACGAGAACCTGATC CGGGTCTTCC AGGAGGGGCG GGACATCCAC 1920 ACGGAGACCG CCAGCTGGATGTTCGGCGTC CCCCGGGAGG CCGTGGACCC CCTGATGCGC 1980 CGGGCGGCCA AGACCATCAACTTCGGGGTC CTCTACGGCA TGTCGGCCCA CCGCCTCTCC 2040 CAGGAGCTAG CTAGCCATCCCTTACGAGGA GGCCCAGGCC TTCATTGA 2088 962 base pairs nucleic acid singlelinear DNA (genomic) 11 ATGAATTCGG GGATGCTGCC CCTCTTTGAG CCCAAGGGCCGGGTCCTCCT GGTGGACGGC 60 CACCACCTGG CCTACCGCAC CTTCCACGCC CTGAAGGGCCTCACCACCAG CCGGGGGGAG 120 CCGGTGCAGG CGGTCTACGG CTTCGCCAAG AGCCTCCTCAAGGCCCTCAA GGAGGACGGG 180 GACGCGGTGA TCGTGGTCTT TGACGCCAAG GCCCCCTCCTTCCGCCACGA GGCCTACGGG 240 GGGTACAAGG CGGGCCGGGC CCCCACGCCG GAGGACTTTCCCCGGCAACT CGCCCTCATC 300 AAGGAGCTGG TGGACCTCCT GGGGCTGGCG CGCCTCGAGGTCCCGGGCTA CGAGGCGGAC 360 GACGTCCTGG CCAGCCTGGC CAAGAAGGCG GAAAAGGAGGGCTACGAGGT CCGCATCCTC 420 ACCGCCGACA AAGACCTTTA CCAGCTTCTT TCCGACCGCATCCACGTCCT CCACCCCGAG 480 GGGTACCTCA TCACCCCGGC CTGGCTTTGG GAAAAGTACGGCCTGAGGCC CGACCAGTGG 540 GCCGACTACC GGGCCCTGAC CGGGGACGAG TCCGACAACCTTCCCGGGGT CAAGGGCATC 600 GGGGAGAAGA CGGCGAGGAA GCTTCTGGAG GAGTGGGGGAGCCTGGAAGC CCTCCTCAAG 660 AACCTGGACC GGCTGAAGCC CGCCATCCGG GAGAAGATCCTGGCCCACAT GGACGATCTG 720 AAGCTCTCCT GGGACCTGGC CAAGGTGCGC ACCGACCTGCCCCTGGAGGT GGACTTCGCC 780 AAAAGGCGGG AGCCCGACCG GGAGAGGCTT AGGGCCTTTCTGGAGAGGCT TGAGTTTGGC 840 AGCCTCCTCC ACGAGTTCGG CCTTCTGGAA AGCCCCAAGTCATGGAGGGG GTGTATCCCC 900 TGGCCGTGCC CCTGGAGGTG GAGGTGGGGA TAGGGGAGGACTGGCTCTCC GCCAAGGAGT 960 GA 962 1600 base pairs nucleic acid doublelinear DNA (genomic) 12 ATGGAATTCG GGGATGCTGC CCCTCTTTGA GCCCAAGGGCCGGGTCCTCC TGGTGGACGG 60 CCACCACCTG GCCTACCGCA CCTTCCACGC CCTGAAGGGCCTCACCACCA GCCGGGGGGA 120 GCCGGTGCAG GCGGTCTACG GCTTCGCCAA GAGCCTCCTCAAGGCCCTCA AGGAGGACGG 180 GGACGCGGTG ATCGTGGTCT TTGACGCCAA GGCCCCCTCCTTCCGCCACG AGGCCTACGG 240 GGGGTACAAG GCGGGCCGGG CCCCCACGCC GGAGGACTTTCCCCGGCAAC TCGCCCTCAT 300 CAAGGAGCTG GTGGACCTCC TGGGGCTGGC GCGCCTCGAGGTCCCGGGCT ACGAGGCGGA 360 CGACGTCCTG GCCAGCCTGG CCAAGAAGGC GGAAAAGGAGGGCTACGAGG TCCGCATCCT 420 CACCGCCGAC AAAGACCTTT ACCAGCTCCT TTCCGACCGCATCCACGTCC TCCACCCCGA 480 GGGGTACCTC ATCACCCCGG CCTGGCTTTG GGAAAAGTACGGCCTGAGGC CCGACCAGTG 540 GGCCGACTAC CGGGCCCTGA CCGGGGACGA GTCCGACAACCTTCCCGGGG TCAAGGGCAT 600 CGGGGAGAAG ACGGCGAGGA AGCTTCTGGA GGAGTGGGGGAGCCTGGAAG CCCTCCTCAA 660 GAACCTGGAC CGGCTGAAGC CCGCCATCCG GGAGAAGATCCTGGCCCACA TGGACGATCT 720 GAAGCTCTCC TGGGACCTGG CCAAGGTGCG CACCGACCTGCCCCTGGAGG TGGACTTCGC 780 CAAAAGGCGG GAGCCCGACC GGGAGAGGCT TAGGGCCTTTCTGGAGAGGC TTGAGTTTGG 840 CAGCCTCCTC CACGAGTTCG GCCTTCTGGA AAGCCCCAAGATCCGCCGGG CCTTCATCGC 900 CGAGGAGGGG TGGCTATTGG TGGCCCTGGA CTATAGCCAGATAGAGCTCA GGGTGCTGGC 960 CCACCTCTCC GGCGACGAGA ACCTGATCCG GGTCTTCCAGGAGGGGCGGG ACATCCACAC 1020 GGAGACCGCC AGCTGGATGT TCGGCGTCCC CCGGGAGGCCGTGGACCCCC TGATGCGCCG 1080 GGCGGCCAAG ACCATCAACT TCGGGGTCCT CTACGGCATGTCGGCCCACC GCCTCTCCCA 1140 GGAGCTAGCC ATCCCTTACG AGGAGGCCCA GGCCTTCATTGAGCGCTACT TTCAGAGCTT 1200 CCCCAAGGTG CGGGCCTGGA TTGAGAAGAC CCTGGAGGAGGGCAGGAGGC GGGGGTACGT 1260 GGAGACCCTC TTCGGCCGCC GCCGCTACGT GCCAGACCTAGAGGCCCGGG TGAAGAGCGT 1320 GCGGGAGGCG GCCGAGCGCA TGGCCTTCAA CATGCCCGTCCGGGGCACCG CCGCCGACCT 1380 CATGAAGCTG GCTATGGTGA AGCTCTTCCC CAGGCTGGAGGAAATGGGGG CCAGGATGCT 1440 CCTTCAGGTC CACGACGAGC TGGTCCTCGA GGCCCCAAAAGAGAGGGCGG AGGCCGTGGC 1500 CCGGCTGGCC AAGGAGGTCA TGGAGGGGGT GTATCCCCTGGCCGTGCCCC TGGAGGTGGA 1560 GGTGGGGATA GGGGAGGACT GGCTCTCCGC CAAGGAGTGA1600 36 base pairs nucleic acid single linear DNA (genomic) 13CACGAATTCG GGGATGCTGC CCCTCTTTGA GCCCAA 36 34 base pairs nucleic acidsingle linear DNA (genomic) 14 GTGAGATCTA TCACTCCTTG GCGGAGAGCC AGTC 3491 base pairs nucleic acid single linear DNA (genomic) 15 TAATACGACTCACTATAGGG AGACCGGAAT TCGAGCTCGC CCGGGCGAGC TCGAATTCCG 60 TGTATTCTATAGTGTCACCT AAATCGAATT C 91 20 base pairs nucleic acid single linear DNA(genomic) 16 TAATACGACT CACTATAGGG 20 27 base pairs nucleic acid singlelinear DNA (genomic) 17 GAATTCGATT TAGGTGACAC TATAGAA 27 31 base pairsnucleic acid single linear DNA (genomic) 18 GTAATCATGG TCATAGCTGGTAGCTTGCTA C 31 42 base pairs nucleic acid single linear DNA (genomic)19 GGATCCTCTA GAGTCGACCT GCAGGCATGC CTACCTTGGT AG 42 30 base pairsnucleic acid single linear DNA (genomic) 20 GGATCCTCTA GAGTCGACCTGCAGGCATGC 30 2502 base pairs nucleic acid double linear DNA (genomic)21 ATGAATTCGG GGATGCTGCC CCTCTTTGAG CCCAAGGGCC GGGTCCTCCT GGTGGACGGC 60CACCACCTGG CCTACCGCAC CTTCCACGCC CTGAAGGGCC TCACCACCAG CCGGGGGGAG 120CCGGTGCAGG CGGTCTACGG CTTCGCCAAG AGCCTCCTCA AGGCCCTCAA GGAGGACGGG 180GACGCGGTGA TCGTGGTCTT TGACGCCAAG GCCCCCTCCT TCCGCCACGA GGCCTACGGG 240GGGTACAAGG CGGGCCGGGC CCCCACGCCG GAGGACTTTC CCCGGCAACT CGCCCTCATC 300AAGGAGCTGG TGGACCTCCT GGGGCTGGCG CGCCTCGAGG TCCCGGGCTA CGAGGCGGAC 360GACGTCCTGG CCAGCCTGGC CAAGAAGGCG GAAAAGGAGG GCTACGAGGT CCGCATCCTC 420ACCGCCGACA AAGACCTTTA CCAGCTCCTT TCCGACCGCA TCCACGTCCT CCACCCCGAG 480GGGTACCTCA TCACCCCGGC CTGGCTTTGG GAAAAGTACG GCCTGAGGCC CGACCAGTGG 540GCCGACTACC GGGCCCTGAC CGGGGACGAG TCCGACAACC TTCCCGGGGT CAAGGGCATC 600GGGGAGAAGA CGGCGAGGAA GCTTCTGGAG GAGTGGGGGA GCCTGGAAGC CCTCCTCAAG 660AACCTGGACC GGCTGAAGCC CGCCATCCGG GAGAAGATCC TGGCCCACAT GGACGATCTG 720AAGCTCTCCT GGGACCTGGC CAAGGTGCGC ACCGACCTGC CCCTGGAGGT GGACTTCGCC 780AAAAGGCGGG AGCCCGACCG GGAGAGGCTT AGGGCCTTTC TGGAGAGGCT TGAGTTTGGC 840AGCCTCCTCC ACGAGTTCGG CCTTCTGGAA AGCCCCAAGG CCCTGGAGGA GGCCCCCTGG 900CCCCCGCCGG AAGGGGCCTT CGTGGGCTTT GTGCTTTCCC GCAAGGAGCC CATGTGGGCC 960GATCTTCTGG CCCTGGCCGC CGCCAGGGGG GGCCGGGTCC ACCGGGCCCC CGAGCCTTAT 1020AAAGCCCTCA GGGACCTGAA GGAGGCGCGG GGGCTTCTCG CCAAAGACCT GAGCGTTCTG 1080GCCCTGAGGG AAGGCCTTGG CCTCCCGCCC GGCGACGACC CCATGCTCCT CGCCTACCTC 1140CTGGACCCTT CCAACACCAC CCCCGAGGGG GTGGCCCGGC GCTACGGCGG GGAGTGGACG 1200GAGGAGGCGG GGGAGCGGGC CGCCCTTTCC GAGAGGCTCT TCGCCAACCT GTGGGGGAGG 1260CTTGAGGGGG AGGAGAGGCT CCTTTGGCTT TACCGGGAGG TGGAGAGGCC CCTTTCCGCT 1320GTCCTGGCCC ACATGGAGGC CACGGGGGTG CGCCTGGACG TGGCCTATCT CAGGGCCTTG 1380TCCCTGGAGG TGGCCGGGGA GATCGCCCGC CTCGAGGCCG AGGTCTTCCG CCTGGCCGGC 1440CACCCCTTCA ACCTCAACTC CCGGGACCAG CTGGAAAGGG TCCTCTTTGA CGAGCTAGGG 1500CTTCCCGCCA TCGGCAAGAC GGAGAAGACC GGCAAGCGCT CCACCAGCGC CGCCGTCCTG 1560GAGGCCCTCC GCGAGGCCCA CCCCATCGTG GAGAAGATCC TGCAGTACCG GGAGCTCACC 1620AAGCTGAAGA GCACCTACAT TGACCCCTTG CCGGACCTCA TCCACCCCAG GACGGGCCGC 1680CTCCACACCC GCTTCAACCA GACGGCCACG GCCACGGGCA GGCTAAGTAG CTCCGATCCC 1740AACCTCCAGA ACATCCCCGT CCGCACCCCG CTTGGGCAGA GGATCCGCCG GGCCTTCATC 1800GCCGAGGAGG GGTGGCTATT GGTGGCCCTG GACTATAGCC AGATAGAGCT CAGGGTGCTG 1860GCCCACCTCT CCGGCGACGA GAACCTGATC CGGGTCTTCC AGGAGGGGCG GGACATCCAC 1920ACGGAGACCG CCAGCTGGAT GTTCGGCGTC CCCCGGGAGG CCGTGGACCC CCTGATGCGC 1980CGGGCGGCCA AGACCATCAA CTTCGGGGTC CTCTACGGCA TGTCGGCCCA CCGCCTCTCC 2040CAGGAGCTAG CCATCCCTTA CGAGGAGGCC CAGGCCTTCA TTGAGCGCTA CTTTCAGAGC 2100TTCCCCAAGG TGCGGGCCTG GATTGAGAAG ACCCTGGAGG AGGGCAGGAG GCGGGGGTAC 2160GTGGAGACCC TCTTCGGCCG CCGCCGCTAC GTGCCAGACC TAGAGGCCCG GGTGAAGAGC 2220GTGCGGGAGG CGGCCGAGCG CATGGCCTTC AACATGCCCG TCCGGGGCAC CGCCGCCGAC 2280CTCATGAAGC TGGCTATGGT GAAGCTCTTC CCCAGGCTGG AGGAAATGGG GGCCAGGATG 2340CTCCTTCAGG TCCACGACGA GCTGGTCCTC GAGGCCCCAA AAGAGAGGGC GGAGGCCGTG 2400GCCCGGCTGG CCAAGGAGGT CATGGAGGGG GTGTATCCCC TGGCCGTGCC CCTGGAGGTG 2460GAGGTGGGGA TAGGGGAGGA CTGGCTCTCC GCCAAGGAGT GA 2502 19 base pairsnucleic acid single linear DNA (genomic) 22 GATTTAGGTG ACACTATAG 19 72base pairs nucleic acid single linear DNA (genomic) 23 CGGACGAACAAGCGAGACAG CGACACAGGT ACCACATGGT ACAAGAGGCA AGAGAGACGA 60 CACAGCAGAA AC72 70 base pairs nucleic acid single linear DNA (genomic) 24 GTTTCTGCTGTGTCGTCTCT CTTGCCTCTT GTACCATGTG GTACCTGTGT CGCTGTCTCG 60 CTTGTTCGTC 7020 base pairs nucleic acid single linear DNA (genomic) 25 GACGAACAAGCGAGACAGCG 20 24 base pairs nucleic acid single linear DNA (genomic) 26GTTTCTGCTG TGTCGTCTCT CTTG 24 46 base pairs nucleic acid single linearDNA (genomic) 27 CCTCTTGTAC CATGTGGTAC CTGTGTCGCT GTCTCGCTTG TTCGTC 4650 base pairs nucleic acid single linear DNA (genomic) 28 ACACAGGTACCACATGGTAC AAGAGGCAAG AGAGACGACA CAGCAGAAAC 50 15 amino acids amino acidsingle unknown protein 29 Met Ala Ser Met Thr Gly Gly Gln Gln Met GlyArg Ile Asn Ser 1 5 10 15 969 base pairs nucleic acid single linear DNA(genomic) 30 ATGGCTAGCA TGACTGGTGG ACAGCAAATG GGTCGGATCA ATTCGGGGATGCTGCCCCTC 60 TTTGAGCCCA AGGGCCGGGT CCTCCTGGTG GACGGCCACC ACCTGGCCTACCGCACCTTC 120 CACGCCCTGA AGGGCCTCAC CACCAGCCGG GGGGAGCCGG TGCAGGCGGTCTACGGCTTC 180 GCCAAGAGCC TCCTCAAGGC CCTCAAGGAG GACGGGGACG CGGTGATCGTGGTCTTTGAC 240 GCCAAGGCCC CCTCCTTCCG CCACGAGGCC TACGGGGGGT ACAAGGCGGGCCGGGCCCCC 300 ACGCCGGAGG ACTTTCCCCG GCAACTCGCC CTCATCAAGG AGCTGGTGGACCTCCTGGGG 360 CTGGCGCGCC TCGAGGTCCC GGGCTACGAG GCGGACGACG TCCTGGCCAGCCTGGCCAAG 420 AAGGCGGAAA AGGAGGGCTA CGAGGTCCGC ATCCTCACCG CCGACAAAGACCTTTACCAG 480 CTTCTTTCCG ACCGCATCCA CGTCCTCCAC CCCGAGGGGT ACCTCATCACCCCGGCCTGG 540 CTTTGGGAAA AGTACGGCCT GAGGCCCGAC CAGTGGGCCG ACTACCGGGCCCTGACCGGG 600 GACGAGTCCG ACAACCTTCC CGGGGTCAAG GGCATCGGGG AGAAGACGGCGAGGAAGCTT 660 CTGGAGGAGT GGGGGAGCCT GGAAGCCCTC CTCAAGAACC TGGACCGGCTGAAGCCCGCC 720 ATCCGGGAGA AGATCCTGGC CCACATGGAC GATCTGAAGC TCTCCTGGGACCTGGCCAAG 780 GTGCGCACCG ACCTGCCCCT GGAGGTGGAC TTCGCCAAAA GGCGGGAGCCCGACCGGGAG 840 AGGCTTAGGG CCTTTCTGGA GAGGCTTGAG TTTGGCAGCC TCCTCCACGAGTTCGGCCTT 900 CTGGAAAGCC CCAAGTCATG GAGGGGGTGT ATCCCCTGGC CGTGCCCCTGGAGGTGGAGG 960 TGGGGATAG 969 948 base pairs nucleic acid single linearDNA (genomic) 31 ATGGCTAGCA TGACTGGTGG ACAGCAAATG GGTCGGATCA ATTCGGGGATGCTGCCCCTC 60 TTTGAGCCCA AGGGCCGGGT CCTCCTGGTG GACGGCCACC ACCTGGCCTACCGCACCTTC 120 CACGCCCTGA AGGGCCTCAC CACCAGCCGG GGGGAGCCGG TGCAGGCGGTCTACGGCTTC 180 GCCAAGAGCC TCCTCAAGGC CCTCAAGGAG GACGGGGACG CGGTGATCGTGGTCTTTGAC 240 GCCAAGGCCC CCTCCTTCCG CCACGAGGCC TACGGGGGGT ACAAGGCGGGCCGGGCCCCC 300 ACGCCGGAGG ACTTTCCCCG GCAACTCGCC CTCATCAAGG AGCTGGTGGACCTCCTGGGG 360 CTGGCGCGCC TCGAGGTCCC GGGCTACGAG GCGGACGACG TCCTGGCCAGCCTGGCCAAG 420 AAGGCGGAAA AGGAGGGCTA CGAGGTCCGC ATCCTCACCG CCGACAAAGACCTTTACCAG 480 CTTCTTTCCG ACCGCATCCA CGTCCTCCAC CCCGAGGGGT ACCTCATCACCCCGGCCTGG 540 CTTTGGGAAA AGTACGGCCT GAGGCCCGAC CAGTGGGCCG ACTACCGGGCCCTGACCGGG 600 GACGAGTCCG ACAACCTTCC CGGGGTCAAG GGCATCGGGG AGAAGACGGCGAGGAAGCTT 660 CTGGAGGAGT GGGGGAGCCT GGAAGCCCTC CTCAAGAACC TGGACCGGCTGAAGCCCGCC 720 ATCCGGGAGA AGATCCTGGC CCACATGGAC GATCTGAAGC TCTCCTGGGACCTGGCCAAG 780 GTGCGCACCG ACCTGCCCCT GGAGGTGGAC TTCGCCAAAA GGCGGGAGCCCGACCGGGAG 840 AGGCTTAGGG CCTTTCTGGA GAGGCTTGAG TTTGGCAGCC TCCTCCACGAGTTCGGCCTT 900 CTGGAAAGCC CCAAGGCCGC ACTCGAGCAC CACCACCACC ACCACTGA 948206 base pairs nucleic acid single linear DNA (genomic) 32 CGCCAGGGTTTTCCCAGTCA CGACGTTGTA AAACGACGGC CAGTGAATTG TAATACGACT 60 CACTATAGGGCGAATTCGAG CTCGGTACCC GGGGATCCTC TAGAGTCGAC CTGCAGGCAT 120 GCAAGCTTGAGTATTCTATA GTGTCACCTA AATAGCTTGG CGTAATCATG GTCATAGCTG 180 TTTCCTGTGTGAAATTGTTA TCCGCT 206 43 base pairs nucleic acid single linear DNA(genomic) 33 TTCTGGGTTC TCTGCTCTCT GGTCGCTGTC TCGCTTGTTC GTC 43 19 basepairs nucleic acid single linear DNA (genomic) 34 GCTGTCTCGC TTGTTCGTC19 20 base pairs nucleic acid single linear DNA (genomic) 35 GACGAACAAGCGAGACAGCG 20 24 base pairs nucleic acid single linear DNA (genomic) 36TTCTGGGTTC TCTGCTCTCT GGTC 24 43 base pairs nucleic acid single linearDNA (genomic) 37 GACGAACAAG CGAGACAGCG ACCAGAGAGC AGAGAACCCA GAA 43 23base pairs nucleic acid single linear DNA (genomic) 38 ACCAGAGAGCAGAGAACCCA GAA 23 21 base pairs nucleic acid single linear DNA (genomic)39 AACAGCTATG ACCATGATTA C 21 60 base pairs nucleic acid single linearDNA (genomic) 40 GTTCTCTGCT CTCTGGTCGC TGTCTCGCTT GTGAAACAAG CGAGACAGCGTGGTCTCTCG 60 15 base pairs nucleic acid single linear DNA (genomic) 41CGAGAGACCA CGCTG 15 52 base pairs nucleic acid single linear DNA(genomic) 42 CCTTTCGCTT TCTTCCCTTC CTTTCTCGCC ACGTTCGCCG GCTTTCCCCG TC52 26 base pairs nucleic acid single linear DNA (genomic) 43 AGAAAGGAAGGGAAGAAAGC GAAAGG 26 21 base pairs nucleic acid single linear DNA(genomic) 44 GACGGGGAAA GCCGGCGAAC G 21 20 base pairs nucleic acidsingle linear DNA (genomic) 45 GAAAGCCGGC GAACGTGGCG 20 21 base pairsnucleic acid single linear DNA (genomic) 46 GGCGAACGTG GCGAGAAAGG A 2142 base pairs nucleic acid single linear DNA (genomic) 47 CCTTTCGCTTTCTTCCCTTC CTTTCTCGCC ACGTTCGCCG GC 42 42 base pairs nucleic acid singlelinear DNA (genomic) 48 CCTTTCGCTC TCTTCCCTTC CTTTCTCGCC ACGTTCGCCG GC42

We claim:
 1. A method of detecting the presence of a target RNA moleculeby detecting non-target cleavage products comprising: a) providing: i) acleavage means, ii) a source of target RNA, said target RNA having afirst region, a second region and a third region, wherein said firstregion is located adjacent to and downstream from said second region andwherein said second region is located adjacent to and downstream fromsaid third region; iii) a first oligonucleotide having a 5′ and a 3′portion wherein said 5′ portion of said first oligonucleotide contains asequence complementary to said second region of said target RNA andwherein said 3′ portion of said first oligonucleotide contains asequence complementary to said third region of said target RNA; iv) asecond oligonucleotide having a 5′ and a 3′ portion wherein said 5′portion of said second oligonucleotide contains a sequence complementaryto said first region of said target RNA and wherein said 3′ portion ofsaid second oligonucleotide contains a sequence complementary to saidsecond region of said target nucleic acid; b) mixing said cleavagemeans, said target RNA, said first oligonucleotide and said secondoligonucleotide to create a reaction mixture under reaction conditionssuch that at least said 3′ portion of said first oligonucleotide isannealed to said target RNA and wherein at least said 5′ portion of saidsecond oligonucleotide is annealed to said target RNA so as to create acleavage structure and wherein cleavage of said cleavage structureoccurs to generate non-target cleavage products; and c) detecting saidnon-target cleavage products.
 2. The method of claim 1 wherein saidreaction conditions comprise a cleavage reaction temperature which isless than the melting temperature of said first oligonucleotide whenannealed to said target RNA and greater than the melting temperature ofsaid 3′ portion of said first oligonucleotide.
 3. The method of claim 1wherein said reaction temperature is between approximately 40 and 65degrees centigrade.
 4. The method of claim 1 wherein said first andsecond oligonucleotides comprise DNA.
 5. The method of claim 1 whereinsaid cleavage means comprises a thermostable 5′ nuclease.
 6. The methodof claim 5 wherein a portion of the amino acid sequence of said nucleaseis homologous to a portion of the amino acid sequence of a thermostableDNA polymerase derived from a thermophilic organism.
 7. The method ofclaim 6 wherein said organism is selected from the group consisting ofThermus aquaticus, Thermus flavus and Thermus thermophilus.
 8. Themethod of claim 7 wherein said nuclease is encoded by a DNA sequenceselected from the group consisting of SEQ ID NOS:1-3, 9, 10, 12, 21, 30and
 31. 9. The method of claim 1 wherein said first oligonucleotide iscompletely complementary to said target RNA and wherein said secondoligonucleotide is completely complementary to said target RNA.
 10. Themethod of claim 1 wherein said first oligonucleotide is partiallycomplementary to said target RNA.
 11. The method of claim 1 wherein saidsecond oligonucleotide is partially complementary to said target RNA.12. The method of claim 1 wherein said detection of said non-targetcleavage products comprises electrophoretic separation of the productsof said reaction followed by visualization of said separated non-targetcleavage products.
 13. The method of claim 1 wherein said source oftarget RNA comprises a sample selected from the group comprising blood,saliva, cerebral spinal fluid, pleural fluid, milk, lymph, sputum andsemen.
 14. The method of claim 1 wherein said reaction conditionscomprise providing a source of divalent cations.
 15. The method of claim14 wherein said divalent cation is selected from the group comprisingMn²⁺ and Mg²⁺ ions.
 16. A method of separating nucleic acid molecules,comprising: a) providing: i) a charge-balanced oligonucleotide and ii) areactant; b) mixing said charge-balanced oligonucleotide with saidreactant to create a reaction mixture under conditions such that acharge-unbalanced oligonucleotide is produced; and c) separating saidcharge-unbalanced oligonucleotide from said reaction mixture.
 17. Themethod of claim 16, wherein said reactant comprises a cleavage means.18. The method of claim 17, wherein said cleavage means is anendonuclease.
 19. The method of claim 17, wherein said cleavage means isan exonuclease.
 20. The method of claim 16, wherein said reactantcomprises a polymerization means.
 21. The method of claim 16, whereinsaid reactant comprises a ligation means.
 22. The method of claim 16,wherein said charge-balanced oligonucleotide comprises a label.
 23. Themethod of claim 16, wherein said charge-balanced oligonucleotidecomprises one or more phosphonate groups.
 24. The method of claim 16,wherein said charge-balanced oligonucleotide has a net neutral chargeand said charge-unbalanced oligonucleotide has a net positive charge.25. The method of claim 16, wherein said charge-balanced oligonucleotidehas a net neutral charge and said charge-unbalanced oligonucleotide hasa net negative charge.
 26. The method of claim 16, wherein saidcharge-balanced oligonucleotide has a net negative charge and saidcharge-unbalanced oligonucleotide has a net positive charge.
 27. Themethod of claim 16, wherein said charge-balanced oligonucleotide has anet negative charge and said charge-unbalanced oligonucleotide has a netneutral charge.
 28. The method of claim 16, wherein said charge-balancedoligonucleotide has a net positive charge and said charge-unbalancedoligonucleotide has a net neutral charge.
 29. The method of claim 16,wherein said charge-balanced oligonucleotide has a net positive chargeand said charge-unbalanced oligonucleotide has a net negative charge.30. The method of claim 17, wherein said charge-balanced oligonucleotidecomprises DNA containing one or more positively charged adducts.
 31. Themethod of claim 30, wherein said cleavage means removes one or morenucleotides from said charge-balanced oligonucleotide to produce saidcharge-unbalanced oligonucleotide, wherein said charge-unbalancedoligonucleotide has a net positive charge.
 32. The method of claim 30,wherein said cleavage means removes one or more nucleotides from saidcharge-balanced oligonucleotide to produce said charge-unbalancedoligonucleotide, wherein said charge-unbalanced oligonucleotide has anet neutral charge.
 33. The method of claim 30, wherein said cleavagemeans removes one or more nucleotides from said charge-balancedoligonucleotide to produce said charge-unbalanced oligonucleotide,wherein said charge-unbalanced oligonucleotide has a net negativecharge.
 34. The method of claim 17, wherein said charge-balancedoligonucleotide comprises DNA containing one or more negatively chargedadducts.
 35. The method of claim 34, wherein said cleavage means removesone or more nucleotides from said charge-balanced oligonucleotide toproduce said charge-unbalanced oligonucleotide, wherein saidcharge-unbalanced oligonucleotide has a net negative charge.
 36. Themethod of claim 34, wherein said cleavage means removes one or morenucleotides from said charge-balanced oligonucleotide to produce saidcharge-unbalanced oligonucleotide, wherein said charge-unbalancedoligonucleotide has a net neutral charge.
 37. The method of claim 34,wherein said cleavage means removes one or more nucleotides from saidcharge-balanced oligonucleotide to produce said charge-unbalancedoligonucleotide, wherein said charge-unbalanced oligonucleotide has anet negative charge.
 38. The method of claim 30, wherein said one ormore positively charged adducts are selected from the group consistingof indodicarbocyanine dye amidites, amino-substituted nucleotides,ethidium bromide, ethidium homodimer, (1,3-propanediamino)propidium,(diethylenetriamino)propidium, thiazole orange,(N-N′-tetramethyl-1,3-propanediamino)propyl thiazole orange,(N-N′-tetramethyl-1,2-ethanediamino)propyl thiazole orange, thiazoleorange-thiazole orange homodimer (TOTO), thiazole orande-thiazole blueheterodimer (TOTAB), thiazole orange-ethidium heterodimer 1 (TOED1),thiazole orange-ethidium heterodimer 2 (TOED2) and florescien-ethidiumheterodimer (FED).
 39. The method of claim 16, wherein said separatingcomprises subjecting said reaction mixture to an electrical fieldcomprising a positive pole and a negative pole under conditions suchthat said charge-unbalanced oligonucleotide migrates toward saidpositive pole.
 40. The method of claim 16, wherein said separatingcomprises subjecting said reaction mixture to an electrical fieldcomprising a positive pole and a negative pole under conditions suchthat said charge-unbalanced oligonucleotide migrates toward saidnegative pole.
 41. The method of claim 39 further comprising detectingthe presence of said separated charge-unbalanced oligonucleotide.
 42. Amethod of detecting cleaved nucleic molecules, comprising: a) providing:i) a homogeneous plurality of charge-balanced oligonucleotides; ii) asample suspected of containing a target nucleic acid having a sequencecomprising a first region complementary to said charge-balancedoligonucleotide; iii) a cleavage means; and iv) a reaction vessel; b)adding to said vessel, in any order, said sample, said charge-balancedoligonucleotides and said cleavage means to create a reaction mixtureunder conditions such that a portion of said charge-balancedoligonucleotides binds to said complementary target nucleic acid tocreate a bound population, and such that said cleavage means cleaves atleast a portion of said bound population of charge-balancedoligonucleotides to produce a population of unbound, charge-unbalancedoligonucleotides; and c) separating said unbound, charge-unbalancedoligonucleotides from sid reaction mixture.
 43. The method of claim 42further comprising providing a homogeneous plurality of oligonucleotidescomplementary to a second region of said target nucleic acid, whereinsaid oligonucleotides are capable of binding to said target nucleic acidupstream of said charge-balanced oligonucleotides.
 44. The method ofclaim 43, wherein said first and said second region of said targetnucleic acid share a region of overlap.
 45. The method of claim 42,wherein said cleavage means comprises a thermostable 5′ nuclease. 46.The method of claim 45 wherein a portion of the amino acid sequence ofsaid nuclease is homologous to a portion of the amino acid sequence of athermostable DNA polymerase derived from a thermophilic organism. 47.The method of claim 46 wherein said organism is selected from the groupconsisting of Thermus aquaticus, Thermus flavus and Thermusthermophilus.
 48. The method of claim 47 wherein said nuclease isencoded by a DNA sequence selected from the group consisting of SEQ IDNOS:1-3, 9, 10, 12, 21, 30 and
 31. 49. The method of claim 42, whereinsaid target nucleic acid comprises single-stranded DNA.
 50. The methodof claim 42 wherein said target nucleic acid comprises double-strandedDNA and prior to the addition of said cleavage means said reactionmixture is treated such that said double-stranded DNA is renderedsubstantially single-stranded.
 51. The method of claim 50 wherein saidtreatment to render said double-stranded DNA is rendered substantiallysingle-stranded by increasing the temperature.
 52. The method of claim42 wherein said target nucleic acid comprises RNA.